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Jochen K. Lennerz Jonathan B. Hurov Lynn S. White Katherine T. Lewandowski Julie L. Prior G. James Planer Robert W. Gereau IV David Piwnica-Worms Robert E. Schmidt Helen Piwnica-Worms 《Molecular and cellular biology》2010,30(21):5043-5056
Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.Cellular polarity is a fundamental principle in biology (6, 36, 62). The prototypical protein kinase originally identified as a regulator of polarity was termed partitioning defective (Par-1) due to early embryonic defects in Caenorhabditis elegans (52). Subsequent studies revealed that Par-1 is required for cellular polarity in worms, flies, frogs, and mammals (4, 17, 58, 63, 65, 71, 89). An integral role for Par-1 kinases in multiple signaling pathways has also been established, and although not formally addressed, multifunctionality for individual Par-1 family members is implied in reviews of the list of recognized upstream regulators and downstream substrates (Table (Table1).1). Interestingly, for many Par-1 substrates the phosphorylated residues generate 14-3-3 binding sites (25, 28, 37, 50, 59, 61, 68, 69, 78, 95, 101, 103). 14-3-3 binding in turn modulates both nuclear/cytoplasmic as well as cytoplasmic/membrane shuttling of target proteins, thus allowing Par-1 activity to establish intracellular spatial organization (15, 101). The phosphorylation of Par-1 itself promotes 14-3-3 binding, thereby regulating its subcellular localization (37, 59, 101).
Open in a separate windowaLKB1 also is known as Par-4; MARKK also is known as Ste20-like; (−), inhibitory/negative regulation has been shown; GPCR, G protein-coupled receptors. MARKK is highly homologous to TAO-1 (thousand-and-one amino acid kinase) (46).The mammalian Par-1 family contains four members (Table (Table2).2). Physiological functions of the Par-1b kinase have been studied using targeted gene knockout approaches in mice (9, 44). Two independently derived mouse lines null for Par-1b have implicated this protein kinase in diverse physiological processes, including fertility (9), immune system homeostasis (44), learning and memory (86), the positioning of nuclei in pancreatic beta cells (35, 38), and growth and metabolism (43).
TABLE 1.
Multifunctionality of Par-1 polarity kinase pathwaysaRegulator or substrate | Function | Reference(s) |
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Regulators (upstream function) | ||
LKB1 | Wnt signaling, Peutz-Jeghers syndrome, insulin signal transduction, pattern formation | 2, 63, 93 |
TAO1 | MEK3/p38 stress-responsive mitogen-activated protein kinase (MAPK) pathway | 46 |
MARKK | Nerve growth factor signaling in neurite development and differentiation | 98 |
aPKC | Ca2+/DAG-independent signal transduction, cell polarity, glucose metabolism | 14, 37, 40, 45, 59, 75, 95 |
nPKC/PKD | DAG-dependent, Ca2+-independent signal transduction (GPCR) | 101 |
PAR-3/PAR-6/aPKC | (−); regulates Par-1, assembly of microtubules, axon-dendrite specification | 19 |
GSK3β | (−); tau phosphorylation, Alzheimer''s dementia, energy metabolism, body patterning | 54, 97 |
Pim-1 oncogene | (−); G2/M checkpoint, effector of cytokine signaling and Jak/STAT(3/5) | 5 |
CaMKI | (−); Ca2+-dependent signal transduction, neuronal differentiation | 99 |
Substrates (downstream function) | ||
Cdc25C | Regulation of mitotic entry by activation of the cdc2-cyclin B complex | 25, 72, 78, 103 |
Class II HDAC | Control of gene expression and master regulator of subcellular trafficking | 28, 50 |
CRTC2/TORC2 | Gluconeogenesis regulator via LKB1/AMPK/TORC2 signaling, PPARγ1a coactivator | 49 |
Dlg/PSD-95 | Synaptogenesis and neuromuscular junction, tumor suppressor (102) | 104 |
Disheveled | Wnt signaling, translocation of Dsh from cytoplasmic vesicles to cortex | 73, 94 |
KSR1 | Regulation of the Ras-MAPK pathway | 68, 69 |
MAP2/4/TAU | Dynamic instability (67, 83) of microtubules, Alzheimer''s dementia (30) | 11, 31-33, 47, 70, 96 |
Mib/Notch | Mind bomb (Mib degradation and repression of Notch signaling results in neurogenesis) | 57, 74, 81 |
Par3/OSKAR/Lgl | Cytoplasmic protein segregation, cell polarity, and asymmetric cell division | 7, 10 |
Pkp2 | Desmosome assembly and organization; nuclear shuttling | 68, 69 |
PTPH1 | Linkage between Ser/Thr and Tyr phosphorylation-dependent signaling | 103 |
Rab11-FIP | Regulation of endocytosis (23), trafficking of E-cadherin (64) | 34 |
TABLE 2.
Terminology and localization of mammalian Par-1 family membersOpen in a separate windowaPar should not to be confused with protease-activated receptor 1 (PAR1 [29]); C-TAK1, Cdc twenty-five C-associated kinase 1; MARK, microtubule affinity regulating kinase; MARKL, MAP/microtubule affinity-regulating kinase-like 1.bBasolateral to a lesser degree than Par-1b (37).cHuman KP78 is asymmetrically localized to the apical surface of epithelial cells (76).dVariant that does not show asymmetric localization in epithelial cells when overexpressed (95).Beyond Par-1b, most information regarding the cell biological functions of the Par-1 kinases comes from studies of Par-1a. Specifically, Par-1a has been implicated in pancreatic (76) and hepatocarcinogenesis (51), as well as colorectal tumors (77), hippocampal function (100), CagA (Helicobacter pylori)-associated epithelial cell polarity disruption (82), and Peutz-Jeghers syndrome (48), although the latter association has been excluded recently (27). As a first step toward determining unique and redundant functions of Par-1 family members, mice disrupted for a second member of the family (Par-1a/MARK3/C-TAK1) were generated. We report that Par-1a−/− mice are viable and develop normally, and adult mice are hypermetabolic, have decreased white and brown adipose tissue mass, and unaltered glucose/insulin handling. However, when challenged by a high-fat diet (HFD), Par-1a−/− mice exhibit resistance to hepatic steatosis, resistance to glucose intolerance, and the delayed onset of obesity relative to that of control littermates. Strikingly, overnight starvation results in a complete depletion of glycogen and lipid stores along with an increase in autophagic vacuoles in the liver of Par-1a−/− but not Par-1b−/− mice. Correspondingly, Par-1a−/− mice develop hypoketotic hypoglycemia. These findings reveal unique metabolic functions of two Par-1 family members. 相似文献6.
Banumathi Sankaran Shilah A. Bonnett Kinjal Shah Scott Gabriel Robert Reddy Paul Schimmel Dmitry A. Rodionov Valérie de Crécy-Lagard John D. Helmann Dirk Iwata-Reuyl Manal A. Swairjo 《Journal of bacteriology》2009,191(22):6936-6949
GTP cyclohydrolase I (GCYH-I) is an essential Zn2+-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn2+-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn2+ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn2+-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn2+-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. (Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. (Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn2+ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH4) pathway (Fig. (Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).
TABLE 1.
Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteriaaOpen in a separate windowaGenes that are preceded by candidate Zur binding sites.bZur-regulated cluster is on the virulence plasmid pLVPK.cExamples of organisms with no folE genes are in boldface type.dZn-dependent regulation of B. subtilis folE2 by Zur was experimentally verified (17).Expression of the Bacillus subtilis folE2 gene, yciA, is controlled by the Zn2+-dependent Zur repressor and is upregulated under Zn2+-limiting conditions (17). This led us to propose that the GCYH-IB family utilizes a metal other than Zn2+ to allow growth in Zn2+-limiting environments, a hypothesis strengthened by the observation that an archaeal ortholog from Methanocaldococcus jannaschii has recently been shown to be Fe2+ dependent (22). To test this hypothesis, we investigated the physiological role of GCYH-IB in B. subtilis, an organism that contains both isozymes, as well as the metal dependence of B. subtilis GCYH-IB in vitro. To gain a structural understanding of the metal dependence of GCYH-IB, we determined high-resolution crystal structures of Zn2+- and Mn2+-bound forms of the N. gonorrhoeae ortholog. Notably, although the GCYH-IA and -IB enzymes belong to the tunneling-fold (T-fold) superfamily, there are significant differences in their global and active-site architecture. These studies shed light on the physiological significance of the alternative folate biosynthesis isozymes in bacteria exposed to various metal environments, and offer a structural understanding of the differential metal dependence of GCYH-IA and -IB. 相似文献7.
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Pamela R. Hall Brian Hjelle Hadya Njus Chunyan Ye Virginie Bondu-Hawkins David C. Brown Kathleen A. Kilpatrick Richard S. Larson 《Journal of virology》2009,83(17):8965-8969
Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β3 (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table (Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).
Open in a separate windowaStandard deviations of four experiments are shown in parentheses. Peptide-bearing phage were added at 109 phage/μl.bP values for the pairwise amino acid alignment score of each peptide versus that of integrin β3 were determined using an unpaired Student''s t test. P values considered statistically significant are shown in bold.To determine whether the peptide sequences of any of the identified inhibitory phage showed homology to integrin β3, a known entry receptor for pathogenic hantaviruses (6, 7), we used the Gap program to perform a pairwise amino acid alignment of each peptide versus the extracellular portion of integrin β3 and determined P values for the alignments. Of 45 phage eluted with the anti-Gn antibody, 6B9/F5, 27 of the peptide sequences showed homology to integrin β3 (P < 0.05), and 9 were highly significant (P ≤ 0.0005) (Fig. (Fig.1A).1A). Of the latter, CKFPLNAAC and CSQFPPRLC map to the hybrid domain (Fig. (Fig.1B),1B), which is proximal to the plexin-semaphorin-integrin domain (PSI) containing residue D39, shown to be critical for viral entry in vitro (19). Five sequences (CPSSPFNH, CPKHVLKVC, CNANKPKMC, CQSQTRNHC, and CDQRTTRLC) map to the I-like (or βA) domain near the binding site of ReoPro (2). Finally, CLPTDPIQC maps to the epidermal growth factor 4 (EGF-4) domain, and CSTRAENQC aligns to a portion of β3 untraceable in the crystal structure, specifically the linker region between the hybrid domain and EGF-1. Although this represents a disordered portion of the protein (22), the location of this loop proximal to the PSI domain is worth noting, due to the role of the PSI domain in facilitating viral entry (19). Therefore, 60% of phage eluted with the anti-Gn antibody showed some homology to integrin β3, and those with highly significant P values predominantly mapped to or proximal to regions of known interest in viral entry.Open in a separate windowFIG. 1.Inhibitory peptides identified through phage panning against ANDV show homology to integrin β3. (A) Alignment of phage peptide sequences with P values for integrin β3 pairwise alignment of less than 0.05. Residues comprising the signal peptide, transmembrane, and cytoplasmic domains, which were not included during pairwise alignment, are underlined. Residues 461 to 548, which are missing in the crystal structure, are italicized. Residues involved in the ReoPro binding site are highlighted in green (2). Residue D39 of the PSI domain is highlighted in yellow (19). Peptides are shown above the sequence of integrin β3, with antibody 6C5/D12-eluted sequences shown in blue text and sequences eluted with antibody 6B9/F5 shown in red. Peptide sequences with alignment P values of ≤0.0005 are highlighted in yellow. Percent inhibition of the peptide-bearing phage is shown in parentheses. (B) View of integrin αvβ3 (PDB ID 1U8C [23]). αv is shown in blue ribbon diagram, and β3 is shown in salmon-colored surface representation, with specific domains circled. Residues corresponding to the ReoPro binding site are shown in green, as in panel A, and D39 is shown in yellow. Regions corresponding to 6C5/D12-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 (highlighted in panel A) are shown in blue, and those corresponding to 6B9/F5-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 are shown in red. Alignment of peptide PLASTRT (P value of 0.0040) adjacent to D39 of the PSI domain is shown in magenta. Graphics were prepared using Pymol (DeLano Scientific LLC, San Carlos, CA).Of the 41 peptide-bearing phage eluted with the anti-Gc antibody 6C5/D12, 14 showed sequence homology to integrin β3 (P < 0.05), 4 of which had P values of ≤0.0005 (Fig. (Fig.1A).1A). Of the latter, sequence CTTMTRMTC mapped to the base of the I-like domain (Fig. (Fig.1B),1B), while CHGVYALHC and CRDTTPWWC mapped to the EGF-3 domain. Finally, sequence CTPTMHNHC mapped to the linker region untraceable in the crystal structure. Therefore, in contrast to peptide sequences identified by competition with the anti-Gn antibody, sequences identified by competition with the anti-Gc antibody 6C5/D12 appear to be mostly unrelated to integrin β3.As a low level of pathogenic hantavirus infection can be seen in cells lacking integrin β3, such as CHO cells (19), we asked if any of the identified peptide sequences could represent a previously unidentified receptor. We used the Basic Local Alignment Search Tool to search a current database of human protein sequences for potential alternate receptors represented by these peptides. However, none of the alignments identified proteins that are expressed at the cell surface, eliminating them as potential candidates for alternate viral entry receptors. This suggests that the majority of the peptides identified here likely represent novel sequences for binding ANDV surface glycoproteins.To determine whether synthetic peptides would also block infection, we synthesized cyclic peptides based on the 10 most-potent peptide-bearing phage. These peptides, in the context of phage presentation, showed levels of inhibition ranging from 44 to 72% (Table (Table2).2). When tested by IFA at 1 mM, four of the synthetic peptides showed inhibition levels significantly lower than those of the same peptide presented in the context of phage. This is not surprising, as steric factors due to the size of the phage and the multivalent presentation of peptide in the context of phage may both contribute to infection inhibition (8). However, there was no significant difference in inhibition by synthetic peptide versus peptide-bearing phage for six of the sequences, implying that inhibition in the context of phage was due solely to the nature of the peptide itself and not to steric factors or valency considerations contributed by the phage, which contrasts with our previous results, determined by using phage directed against αvβ3 integrin (10).
Open in a separate windowaStandard deviations of the results of at least four experiments are shown in parentheses.bMean percent inhibition between phage and synthetic peptide differs significantly (P < 0.05).The three most-potent synthetic peptides were examined for their ability to inhibit ANDV entry in a dose-dependent manner. The concentration of each peptide that produces 50% of its maximum potential inhibitory effect was determined. As shown in Fig. Fig.2A,2A, the 50% inhibitory concentration for each of the peptides was in the range of 10 μM, which from our experience is a reasonable potency for a lead compound to take forward for optimization.Open in a separate windowFIG. 2.Activities of synthetic peptides in inhibition of ANDV infection in vitro. (A) Peptides were examined for their ability to block ANDV infection of Vero E6 cells in a dose-dependent manner by IFA. (B) Peptides were tested in parallel for the ability to block infection of Vero E6 cells by ANDV, SNV, HTNV, and PHV. (C) Peptides were tested, singly or in combination, for the ability to block ANDV infection of Vero E6 cells. For all experiments, controls included media, ReoPro at 80 μg/ml, and monoclonal antibodies 6C5/D12 and 6B9/F5 at 5 μg/ml. All peptides were used at 1 mM. Data points represent n = 2 to 6, with error bars showing the standard errors of the means. Statistical analyses were performed on replicate samples using an unpaired Student''s t test.In order to determine the specificity of the three most-potent synthetic cyclic peptides in blocking ANDV, we examined them for inhibition of ANDV infection versus two other pathogenic hantaviruses, SNV and Hantaan virus (HTNV), or the nonpathogenic hantavirus Prospect Hill virus (PHV). As shown in Fig. Fig.2B,2B, ReoPro, which binds integrin β3, showed inhibition of infection by each of the pathogenic hantavirus strains, known to enter cells via β3, but not the nonpathogenic PHV, which enters via integrin β1 (6, 7). In contrast, peptides selected for the ability to bind ANDV were highly specific inhibitors of ANDV versus SNV, HTNV, or PHV. The specificities of peptides eluted by the anti-Gn monoclonal antibody are not surprising, as they are likely due to global differences in the Gn amino acid sequence. Specifically, sequence homologies between ANDV and SNV, HTNV, and PHV are 61%, 36%, and 51%, respectively, for the region corresponding to the immunogen for antibody 6B9/F5. Although homology between the immunogen for antibody 6C5/D12 and the corresponding Gc region of these viruses is somewhat higher (82% with SNV, 63% with HTNV, and 71% with PHV), the possibility that the monoclonal antibody used here recognizes a three-dimensional epitope lends itself to the high specificity of the peptides.The current model for cellular infection by hantaviruses (14) is as follows. Viral binding of the host cell surface target integrin is followed by receptor-mediated endocytosis and endosome acidification. Lowered pH induces conformational changes in Gn and/or Gc, which facilitate membrane fusion and viral release into the cytosol. As there is currently little information available about whether one glycoprotein is dominant in mediating infection, and as neutralizing epitopes have been found on both Gn and Gc glycoproteins (1, 4, 12, 13, 20), we examined whether combining anti-Gn- and anti-Gc-targeted synthetic peptides would lead to an increased infection blockade compared to those for single treatments. As shown in Fig. Fig.2C,2C, the combination of anti-Gn and anti-Gc peptides CMQSAAAHC and CTVGPTRSC resulted in a significant increase in infection inhibition (P = 0.0207 for CMQSAAAHC, and P = 0.0308 for CTVGPTRSC) compared to that resulting from single treatments. Although the high specificity of the peptides for ANDV makes it unlikely that this combination treatment will lead to more cross-reactivity with other pathogenic hantaviruses, this can be determined only by additional testing. Regardless, these data suggest a unique role for each of these viral proteins in the infection process as well as the benefits of targeting multiple viral epitopes for preventing infection.To our knowledge, the peptides reported here are the first identified that directly target ANDV, and this work further illustrates the power of coupling phage display and selective elution techniques in the identification of novel peptide sequences capable of specific protein-protein interactions from a large, random pool of peptide sequences. These novel peptide inhibitors (R. S. Larson, P. R. Hall, H. Njus, and B. Hjelle, U.S. patent application 61/205,211) provide leads for the development of more-potent peptide or nonpeptide organics for therapeutic use against HCPS. 相似文献
TABLE 1.
Peptide-bearing phage eluted from ANDVPhage | % Inhibition (SD)a | P valueb |
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Phage bearing the following peptides eluted with anti-Gn antibody 6B9/F5 | ||
Group 1 (<30% inhibition) | ||
CDQRTTRLC | 8.45 (15.34) | 0.0002 |
CPHDPNHPC | 9.94 (7.72) | 0.333 |
CQSQTRNHC | 11.76 (13.25) | 0.0001 |
CLQDMRQFC | 13.26 (9.92) | 0.0014 |
CLPTDPIQC | 15.70 (14.05) | 0.0005 |
CPDHPFLRC | 16.65 (15.22) | 0.8523 |
CSTRAENQC | 17.56 (16.50) | 0.0004 |
CPSHLDAFC | 18.98 (20.06) | 0.0017 |
CKTGHMRIC | 20.84 (7.47) | 0.0563 |
CVRTPTHHC | 20.89 (27.07) | 0.1483 |
CSGVINTTC | 21.57 (19.61) | 0.0643 |
CPLASTRTC | 21.65 (5.98) | 0.004 |
CSQFPPRLC | 22.19 (8.26) | 0.0004 |
CLLNKQNAC | 22.34 (7.78) | 0.001 |
CKFPLNAAC | 22.89 (6.15) | 0.0001 |
CSLTPHRSC | 23.63 (16.74) | 0.0563 |
CKPWPMYSC | 23.71 (6.68) | 0.0643 |
CLQHDALNC | 24.01 (7.60) | 1 |
CNANKPKMC | 24.67 (11.67) | 0.0004 |
CPKHVLKVC | 25.30 (28.36) | 0.0003 |
CTPDKKSFC | 26.91 (11.15) | 0.399 |
CHGKAALAC | 27.22 (32.53) | 0.005 |
CNLMGNPHC | 28.08 (21.35) | 0.0011 |
CLKNWFQPC | 28.64 (18.49) | 0.0016 |
CKEYGRQMC | 28.76 (29.33) | 0.0362 |
CQPSDPHLC | 29.44 (31.22) | 0.0183 |
CSHLPPNRC | 29.70 (17.37) | 0.0061 |
Group 2 (30-59% inhibition) | ||
CSPLLRTVC | 33.05 (20.26) | 0.0023 |
CHKGHTWNC | 34.17 (12.50) | 0.0795 |
CINASHAHC | 35.62 (13.03) | 0.3193 |
CWPPSSRTC | 36.75 (26.95) | 0.0006 |
CPSSPFNHC | 37.78 (7.11) | 0.0001 |
CEHLSHAAC | 38.47 (7.60) | 0.0115 |
CQDRKTSQC | 38.74 (9.12) | 0.1802 |
CTDVYRPTC | 38.90 (25.03) | 0.006 |
CGEKSAQLC | 39.11 (27.52) | 0.0013 |
CSAAERLNC | 40.13 (6.33) | 0.0033 |
CFRTLEHLC | 42.07 (5.01) | 0.0608 |
CEKLHTASC | 43.60 (27.92) | 0.1684 |
CSLHSHKGC | 45.11 (49.81) | 0.0864 |
CNSHSPVHC | 45.40 (28.80) | 0.0115 |
CMQSAAAHC | 48.88 (44.40) | 0.5794 |
CPAASHPRC | 51.84 (17.09) | 0.1935 |
CKSLGSSQC | 53.90 (13.34) | 0.0145 |
Group 3 (60-79% inhibition) | ||
CPSNVNNIC | 61.11 (25.41) | 0.1245 |
Negative control | 0 (6.15) | |
6B9/F5 (5 μg/ml) | 26.77 (5.33) | |
ReoPro (80 μg/ml) | 79.86 (4.88) | |
Phage bearing the following peptides eluted with anti-Gc antibody 6C5/D12 | ||
Group 1 (<30% inhibition) | ||
CHPGSSSRC | 1.01 (7.03) | 0.0557 |
CSLSPLGRC | 10.56 (13.62) | 0.7895 |
CTARYTQHC | 12.86 (3.83) | 0.3193 |
CHGVYALHC | 12.91 (7.32) | 0.0003 |
CLQHNEREC | 16.79 (13.72) | 0.0958 |
CHPSTHRYC | 17.23 (14.53) | 0.0011 |
CPGNWWSTC | 19.34(9.91) | 0.1483 |
CGMLNWNRC | 19.48 (19.42) | 0.0777 |
CPHTQFWQC | 20.44 (13.65) | 0.0008 |
CTPTMHNHC | 20.92 (11.68) | 0.0001 |
CDQVAGYSC | 21.79 (23.60) | 0.0063 |
CIPMMTEFC | 24.33 (9.28) | 0.2999 |
CERPYSRLC | 24.38 (9.09) | 0.0041 |
CPSLHTREC | 25.06 (22.78) | 0.1202 |
CSPLQIPYC | 26.30 (34.29) | 0.4673 |
CTTMTRMTC (×2) | 29.27 (8.65) | 0.0001 |
Group 2 (30-59% inhibition) | ||
CNKPFSLPC | 30.09 (5.59) | 0.4384 |
CHNLESGTC | 31.63 (26.67) | 0.751 |
CNSVPPYQC | 31.96 (6.51) | 0.0903 |
CSDSWLPRC | 32.95 (28.54) | 0.259 |
CSAPFTKSC | 33.40 (10.64) | 0.0052 |
CEGLPNIDC | 35.63 (19.90) | 0.0853 |
CTSTHTKTC | 36.28 (13.42) | 0.132 |
CLSIHSSVC | 36.40 (16.44) | 0.8981 |
CPWSTQYAC | 36.81 (32.81) | 0.5725 |
CTGSNLPIC | 36.83 (31.64) | 0.0307 |
CSLAPANTC | 39.73 (4.03) | 0.1664 |
CGLKTNPAC | 39.75 (16.98) | 0.2084 |
CRDTTPWWC | 40.08 (18.52) | 0.0004 |
CHTNASPHC | 40.26 (4.77) | 0.5904 |
CTSMAYHHC | 41.89 (8.61) | 0.259 |
CSLSSPRIC | 42.13 (29.75) | 0.2463 |
CVSLEHQNC | 45.54 (6.55) | 0.5065 |
CRVTQTHTC | 46.55 (8.45) | 0.3676 |
CPTTKSNVC | 49.28 (14.00) | 0.3898 |
CSPGPHRVC | 49.50 (42.60) | 0.0115 |
CKSTSNVYC | 51.20 (4.60) | 0.0611 |
CTVGPTRSC | 57.30 (11.31) | 0.0176 |
Group 3 (60-79% inhibition) | ||
CPMSQNPTC | 65.60 (13.49) | 0.014 |
CPKLHPGGC | 71.88 (27.11) | 0.0059 |
Negative control | 0.26 (4.53) | |
6C5/D12 (5 μg/ml) | 22.62 (8.40) | |
ReoPro (80 μg/ml) | 80.02 (76.64) |
TABLE 2.
Synthetic cyclic peptides inhibit ANDV infectionTarget | Sample | % Inhibition bya:
| |
---|---|---|---|
Peptide-bearing phage | Synthetic peptide | ||
Gn | CMQSAAAHC | 48.88 (44.40) | 59.66 (11.17) |
Gc | CTVGPTRSC | 57.30 (11.31) | 46.47 (7.61) |
Gn | CPSNVNNIC | 61.11 (25.41) | 44.14 (10.74) |
Gn | CEKLHTASC | 43.60 (27.92) | 34.87 (9.26) |
Gc | CPKLHPGGC | 71.88 (27.11) | 30.95 (7.73)b |
Gn | CSLHSHKGC | 45.11 (49.81) | 29.79 (9.34) |
Gc | CPMSQNPTC | 65.60 (13.49) | 18.19 (8.55)b |
Gn | CKSLGSSQC | 53.90 (13.34) | 18.10 (7.55)b |
Gn | CNSHSPVHC | 45.40 (28.80) | 15.52 (10.48) |
Gn | CPAASHPRC | 51.84 (17.09) | 0 (10.72)b |
Integrin β3 | ReoPro | 80.10 (7.72) | |
Gn | 6B9/F5 antibody | 42.72 (6.75) | |
Gc | 6C5/D12 antibody | 31.04 (7.81) |
13.
Dione B. Rolim Marcos F. G. Rocha Raimunda S. N. Brilhante Rossana A. Cordeiro Natanael P. Leit?o-Junior Timothy J. J. Inglis José J. C. Sidrim 《Applied and environmental microbiology》2009,75(4):1215-1218
Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. (Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. (Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table (Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).
Open in a separate windowaSites designated with B are in Banabuiú, and sites designated with T are in Tejuçuoca. See the text for details.The data on weather and soil composition were obtained from specialized government institutions, such as FUNCEME, IPECE, and EMBRAPA. The average annual temperature in both municipalities is between 26 and 28°C. In 2007, the annual rainfall in Tejuçuoca was 496.8 mm, and that in Banabuiú was 766.8 mm. There are a range of soil types in both Tejuçuoca and Banabuiú: noncalcic brown, sodic planossolic, red-yellow podzolic, and litholic. In Banabuiú, there are also alluvial and cambisol soils. The characteristic vegetation in both municipalities is caatinga (scrublands).There were isolates of B. pseudomallei in 26 (4.3%) of the 600 samples collected. The bacterium was isolated at a rate (3%) similar to that previously reported (9). The bacterium isolation occurred in both the dry (53.8%) and the rainy (46.2%) seasons. Tejuçuoca represented 76.9% (20/26) of the strains isolated. Four sites in Tejuçuoca (T1, T3, T4, and T5) and three in Banabuiú (B1, B2, and B4) presented isolates of the bacterium (Table (Table1).1). The isolation of the B. pseudomallei strains varied from the surface down to 40 cm. However, 17 of the 26 positive samples (65.3%) were found at depths between 20 and 40 cm (Table (Table1).1). Only two isolates were found at the surface during the dry season.A study in Vietnam (13) and one in Australia (9) reported the presence of B. pseudomallei near the houses of melioidosis patients. In our study, the same thing happened. Site T3 (15/26; 57.6%) was located 290 m from the patient''s house, as reported by the Rolim group (14).B. pseudomallei was isolated from a sheep paddock in Australia, where animals sought shelter below mango and fig trees (17). In our study, the bacterium was isolated at site T5, a goat corral alongside the house where the outbreak occurred in Tejuçuoca. Four sites in places shaded by trees yielded positive samples (30.7%) in both Tejuçuoca (palm trees) and Banabuiú (mango trees). Additionally, B. pseudomallei was isolated on three occasions from a cornfield (site 4B) located alongside the house of the melioidosis patient in Banabuiú.In the main areas of endemicity, the disease is more prevalent in the rainy season (4, 5, 16). The outbreak in Tejuçuoca was related to rainfall (14). Besides the association of cases of the disease with rainfall itself, the isolation of B. pseudomallei in soil and water was also demonstrated during the dry season (12, 15). An Australian study isolated strains from soil and water during the dry and rainy seasons (17). A Thai study also reported B. pseudomallei in the dry season (18). In our study, the isolation of B. pseudomallei took place either at the end of the wet season or in the dry months. Fourteen of the positive samples (53.8%) were collected during the dry season, albeit near a river or reservoir (sites T3 and B4).Physical, biological, and chemical soil features appear to influence the survival of B. pseudomallei (6, 10). In the present study, the soil was classified as litholic with sandy or clayey textures. It is susceptible to erosion, and when there is a lack of water, it is subject to salinization. During the dry season, the clay layer becomes dried, cracked, and very hard. During the rainy season, it becomes soggy and sticky. The isolation of B. pseudomallei in the dry season is possibly related to the capacity for adaptation of this soil, since the extreme conditions of lithosols do not prevent the bacterial growth and survival.It has been shown that B. pseudomallei is more often isolated at depths between 25 and 45 cm (17). In our study, 65.3% of the positive samples were taken at depths between 20 and 40 cm. Moreover, of these 17 samples, 10 (58.8%) were collected during the dry months. Also, unlike in other regions, two positive samples were taken from the surface in the period without rainfall.The rainfall in Tejuçuoca and Banabuiú is generally low, and temperatures do not vary significantly during the year. Therefore, the isolation of B. pseudomallei in these places occurs outside the rainfall, temperature, and moisture conditions observed in other regions of endemicity. Our data thus suggest that peculiar environmental features, such as soil composition, might favor the multiplication of B. pseudomallei in northeast Brazil. 相似文献
TABLE 1.
Distribution of samples with isolates by site and soil depthSitesa and depth (cm) | No. of B. pseudomallei isolates in samples from:
| ||
---|---|---|---|
Banabuiú (n = 300) | Tejuçuoca (n = 300) | Total (n = 600) | |
B1/T1 | 3 | ||
Surface | 2 | ||
10 | |||
20 | 1 | ||
30 | |||
40 | |||
B2/T2 | 1 | ||
Surface | 1 | ||
10 | |||
20 | |||
30 | |||
40 | |||
B3/T3 | 15 | ||
Surface | 2 | ||
10 | 2 | ||
20 | 4 | ||
30 | 3 | ||
40 | 4 | ||
B4/T4 | 5 | ||
Surface | |||
10 | 1 | ||
20 | 1 | ||
30 | 1 | 1 | |
40 | 1 | ||
B5/T5 | 2 | ||
Surface | |||
10 | |||
20 | |||
30 | 2 | ||
40 | |||
Total | 6 | 20 | 26 |
14.
Pablo I. Nikel Andrea M. Giordano Alejandra de Almeida Manuel S. Godoy M. Julia Pettinari 《Applied and environmental microbiology》2010,76(22):7400-7406
The effect of eliminating d-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP+ ratio was higher than the NADH/NAD+ ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP+. In 60-h fed-batch cultures, K24KL reached 41.9 g·liter−1 biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g−1, the highest reported using this substrate.Poly(3-hydroxybutyrate) (PHB) is the best-known and most common polyhydroxyalkanoate (PHA). PHAs are polymers with thermoplastic properties that are totally biodegradable by microorganisms present in most environments and that can be produced from different renewable carbon sources (38). Accumulated as intracellular granules by many bacteria under unfavorable conditions (1, 21), PHAs are carbon and energy reserves and also act as electron sinks, enhancing the fitness and stress resistance of bacteria and contributing to redox balance (12, 30). Escherichia coli offers a well-defined physiological environment for the construction and manipulation of various metabolic pathways to produce different bioproducts, such as PHB, from cost-effective carbon sources.In recent years, a significant increase in the production of biodiesel has caused a sharp fall in the cost of glycerol, the main by-product of biodiesel synthesis. As a result, glycerol has become a very attractive substrate for bacterial fermentations (10), specially for reduced products, such as PHB (36). The E. coli strain used in this work, K24K, carries phaBAC, the structural genes responsible for PHB synthesis, from Azotobacter sp. strain FA8 (23) (Table (Table1).1). The pha genes in K24K are expressed from a chimeric promoter and consequently are not subject to the genetic regulatory systems present in natural PHA producers. Because of this, it can be assumed that regulation of PHA synthesis in the recombinants is restricted by enzyme activity levels, modulated principally by substrate availability. In most natural producers, and also in PHB-producing E. coli recombinants, PHB is synthesized through the condensation of two molecules of acetyl-coenzyme A (acetyl-CoA), catalyzed by an acetoacetyl-CoA transferase or 3-ketothiolase, resulting in acetoacetyl-CoA. This compound is subsequently reduced by an NAD(P)H-dependent acetoacetyl-CoA reductase to R-(−)-3-hydroxybutyryl-CoA, which is then polymerized by a specific PHA synthase (34).
Open in a separate windowaStrain obtained through the E. coli Genetic Stock Center, Yale University, New Haven, CT.bFor oligonucleotides, the ATG codon of ldhA is underlined and the sequences with homology to FRT-kan-FRT in the template plasmid pKD4 (11) are shown in boldface.Cells growing on glycerol are in a more reduced intracellular state than cells grown on glucose under similar conditions of oxygen availability. This has a significant effect on the intracellular redox state, which causes the cells to direct carbon flow toward the synthesis of more-reduced products when glycerol is used than when glucose is used in order to achieve redox balance (31). When metabolic product distribution was analyzed in bioreactor cultures of K24K using glucose or glycerol as the substrate, product distributions with the two substrates were found to be different, as glycerol-grown cultures produced smaller amounts of acetate, lactate, and formate and more ethanol than those grown on glucose. However, PHB production from glycerol was lower than that from glucose, except under conditions of low oxygen availability (13).Manipulations to enhance the synthesis of a metabolic product include several approaches to increase the availability of the substrates needed for its formation or to inhibit competing pathways. The effect of eliminating competing pathways on PHB production from glucose has been investigated through the inactivation of different genes, such as those encoding enzymes participating in the synthesis of acetate (ackA, pta, and poxB) or d-lactate (ldhA). A pta mutant, which produces very little acetate (6), and an frdA ldhA double mutant (40) had increased PHB accumulation from glucose. A recent report using an ackA pta poxB ldhA adhE mutant under microaerobic conditions attained similar results (17). The inactivation of ldhA has also been shown to have an important effect on the metabolic product distribution in recombinant E. coli with glycerol as the carbon source, promoting ethanol synthesis (28). In the present work we analyzed the effect of ldhA inactivation in strain K24K using glycerol as the carbon source, with special emphasis on changes in carbon distribution and in the intracellular redox state, determined through cofactor levels. 相似文献
TABLE 1.
E. coli strains, plasmids, and oligonucleotides used in this studyStrain, plasmid, or oligonucleotide | Relevant characteristicsb | Reference or source |
---|---|---|
E. coli strains | ||
K1060a | F−fadE62 lacI60 tyrT58(AS) fabB5 mel-1 | 29 |
K24 | Same as K1060, carrying pJP24; Apr | 23 |
K24K | Same as K1060, carrying pJP24K; Apr Kmr | 23 |
ALS786a | F− λ−rph-1 ΔldhA::kan; Kmr | 14 |
K24LT | Same as K1060 but ΔldhA::kan by K1060 × P1(ALS786), carrying pJP24; Apr Kmr | This work |
K24KL | Same as K1060 but ΔldhA by allelic replacement, carrying pJP24K; Kmr | This work |
TA3522a | F− λ− Δ(his-gnd)861 hisJo-701 | 2 |
TA3514a | Same as TA3522 but pta-200 | 19 |
TA3522L | Same as TA3522 but ΔldhA::kan by TA3522 × P1(ALS786); Kmr | This work |
TA3514L | Same as TA3514 but ΔldhA::kan by TA3514 × P1(ALS786); Kmr | This work |
Plasmids | ||
pQE32 | Expression vector, ColE1 ori; Apr | Qiagen GmbH, Hilden, Germany |
pJP24 | pQE32 derivative expressing a 4.3-kb BamHI-HindIII insert containing the phaBAC genes from Azotobacter sp. strain FA8 under the control of a T5 promoter/lac operator element; Apr | 23 |
pJP24K | pJP24 derivative; Apr Kmr | 23 |
pCP20 | Helper plasmid used for kan excision; Saccharomyces cerevisiae FLP λ cI857 λ PRrepA(Ts); Apr Cmr | 7 |
Oligonucleotides | ||
ΔldhA-F | 5′-TAT TTT TAG TAG CTT AAA TGT GAT TCA ACA TCA CTG GAG AAA GTC TTA TGG TGT AGG CTG GAG CTG CTT C-3′ | This work |
ΔldhA-R | 5′-CTC CCC TGG AAT GCA GGG GAG CGG CAA GAT TAA ACC AGT TCG TTC GGG CAC ATA TGA ATA TCC TCC TTA G-3′ | This work |
15.
16.
17.
Tatiana N. Stekhanova Andrey V. Mardanov Ekaterina Y. Bezsudnova Vadim M. Gumerov Nikolai V. Ravin Konstantin G. Skryabin Vladimir O. Popov 《Applied and environmental microbiology》2010,76(12):4096-4098
Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.Alcohol dehydrogenases (ADHs; EC 1.1.1.1.) catalyze the interconversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. NAD(P)-dependent ADHs, due to their broad substrate specificity and enantioselectivity, have attracted particular attention as catalysts in industrial processes (5). However, mesophilic ADHs are unstable at high temperatures, sensitive to organic solvents, and often lose activity during immobilization. In this relation, there is a considerable interest in ADHs from extremophilic microorganisms; among them, Archaea are of great interest. The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (11, 12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (3, 4, 14, 16, 19) or long-chain iron-activated ADHs (1, 8, 9). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (10, 18) and only one archaeal aldo-keto reductase also from P. furiosus (11) have been characterized.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a high-temperature oil reservoir capable of growth on complex organic substrates (15). The complete genome sequence of T. sibiricus has been recently determined and annotated (13). Several ADHs are encoded by the T. sibiricus genome, including three short-chain ADHs (Tsib_0319, Tsib_0703, and Tsib_1998) (13). In this report, we describe the cloning and expression of the Tsib_0319 gene from T. sibiricus and the purification and the biochemical characterization of its product, the thermostable short-chain ADH (TsAdh319).The Tsib_0319 gene encodes a protein with a size of 234 amino acids and the calculated molecular mass of 26.2 kDa. TsAdh319 has an 85% degree of sequence identity with short-chain ADH from P. furiosus (AdhA; PF_0074) (18). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs. The gene flanked by the XhoI and BamHI sites was PCR amplified using two primers (sense primer, 5′-GTTCTCGAGATGAAGGTTGCTGTGATAACAGGG-3′, and antisense primer, 5′-GCTGGATCCTCAGTATTCTGGTCTCTGGTAGACGG-3′) and cloned into the pET-15b vector. TsAdh319 was overexpressed, with an N-terminal His6 tag in Escherichia coli Rosetta-gami (DE3) and purified to homogeneity by metallochelating chromatography (Hi-Trap chelating HP column; GE Healthcare) followed by gel filtration on Superdex 200 10/300 GL column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl. The homogeneity and the correspondence to the calculated molecular mass of 28.7 kDa were verified by SDS-PAGE (7). The molecular mass of native TsAdh319 was 56 to 60 kDa, which confirmed the dimeric structure in solution.The standard ADH activity measurement was made spectrophotometrically at the optimal pH by following either the reduction of NADP (in 50 mM Gly-NaOH buffer; pH 10.5) or the oxidation of NADPH (in 0.1 M sodium phosphate buffer; pH 7.5) at 340 nm at 60°C. The enzyme exhibited a strong preference for NADP(H) and broad substrate specificity (Table (Table1).1). The highest oxidation rates were found with pentoses d-arabinose (2.0 U mg−1) and d-xylose (2.46 U mg−1), and the highest reduction rates were found with dimethylglyoxal (5.9 U mg−1) and pyruvaldehyde (2.2 U mg−1). The enzyme did not reduce sugars which were good substrates for the oxidation reaction. The kinetic parameters of TsAdh319 determined for the preferred substrates are shown in Table Table2.2. The enantioselectivity of the enzyme was estimated by measuring the conversion rates of 2-butanol enantiomers. TsAdh319 showed an evident preference, >2-fold, for (S)-2-butanol over (RS)-2-butanol. The enzyme stereoselectivity is confirmed by the preferred oxidation of d-arabinose over l-arabinose (Table (Table1).1). The fact that TsAdh319 is metal independent was supported by the absence of a significant effect of TsAdh319 preincubation with 10 mM Me2+ for 30 min before measuring the activity in the presence of 1 mM Me2+ or EDTA (Table (Table3).3). TsAdh319 also exhibited a halophilic property, so the enzyme activity increased in the presence of NaCl and KCl and the activation was maintained even at concentration of 4 M and 3 M, respectively (Table (Table33).
Open in a separate windowaSubstrates were present in 250 mM or 50 mM (*) concentrations.bRelative rates, measured under standard conditions, were calculated by defining the activity for 2-propanol as 100%, which corresponds to 1.0 U mg−1. Data are averages from triplicate experiments.cRelative rates, measured under standard conditions, were calculated by defining the activity for pyruvaldehyde as 100%, which corresponds to 2.2 U mg−1. Data are averages from triplicate experiments.
Open in a separate windowaActivity was measured under standard conditions with 2-propanol. Data are averages from triplicate experiments.bActivity was measured under standard conditions with pyruvaldehyde. Data are averages from triplicate experiments.
Open in a separate windowaThe activity was measured under standard conditions with 2-propanol; relative rates were calculated by defining the activity without salts as 100%, which corresponds to 0.9 U mg−1. Data are averages from duplicate experiments.The most essential distinctions of TsAdh319 are the thermophilicity and high thermostability of the enzyme. The optimum temperature for the 2-propanol oxidation catalyzed by TsAdh319 was not achieved. The initial reaction rate of oxidation increased up to 100°C (Fig. (Fig.1).1). The Arrhenius plot is a straight line, typical of a single rate-limited thermally activated process, but there is no obvious transition point due to the temperature-dependent conformational changes of the protein molecule. The activation energy for the oxidation of 2-propanol was estimated at 84.0 ± 5.8 kJ·mol−1. The thermostability of TsAdh319 was calculated from residual TsAdh319 activity after preincubation of 0.4 mg/ml enzyme solution in 50 mM Tris-HCl buffer (pH 7.5) containing 200 mM NaCl at 70, 80, 90, or 100°C. The preincubation at 70°C or 80°C for 1.5 h did not cause a decrease in the TsAdh319 activity, but provoked slight activation. The residual TsAdh319 activities began to decrease after 2 h of preincubation at 70°C or 80°C and were 10% and 15% down from the control, respectively. The determined half-life values of TsAdh319 were 2 h at 90°C and 1 h at 100°C.Open in a separate windowFIG. 1.Temperature dependence of the initial rate of the 2-propanol reduction by TsAdh319. The reaction was initiated by enzyme addition to a prewarmed 2-propanol-NADP mixture. The inset shows the Arrhenius plot of the same data.Protein thermostability often correlates with such important biotechnological properties as increased solvent tolerance (2). We tested the influence of organic solvents at a high concentration (50% [vol/vol]) on TsAdh319 by using either preincubation of the enzyme at a concentration of 0.2 mg/ml with solvents for 4 h at 55°C or solvent addition into the reaction mixture to distinguish the effect of solvent on the protein stability and on the enzyme activity. TsAdh319 showed significant solvent tolerance in both cases (Table (Table4),4), and the effects of solvents could be modulated by salts, acting apparently as molecular lyoprotectants (17). Furthermore, TsAdh319 maintained 57% of its activity in 25% (vol/vol) 2-propanol, which could be used as the cosubstrate in cofactor regeneration (6).
Open in a separate windowaThe activity measured at the standard condition with 2-propanol as a substrate. Data are averages from triplicate experiments.bPreincubation for 4 h at 55°C in the presence of 50% (vol/vol) of solvent prior the activity assay.cWithout preincubation, solvent addition to the reaction mixture up to 50% (vol/vol) or using the buffer saturated by a solvent (*).dDMSO, dimethyl sulfoxide.eDMFA, dimethylformamide.From all the aforesaid we may suppose TsAdh319 or its improved variant to be interesting both for the investigation of structural features of protein tolerance and for biotechnological applications. 相似文献
TABLE 1.
Substrate specificity of TsAdh319Substratea | Relative activity (%) |
---|---|
Oxidation reactionb | |
Methanol | 0 |
2-Methoxyethanol | 0 |
Ethanol | 36 |
1-Butanol | 80 |
2-Propanol | 100 |
(RS)-(±)-2-Butanol | 86 |
(S)-(+)-2-Butanol | 196 |
2-Pentanol | 67 |
1-Phenylmethanol | 180 |
1.3-Butanediol | 91 |
Ethyleneglycol | 0 |
Glycerol | 16 |
d-Arabinose* | 200 |
l-Arabinose* | 17 |
d-Xylose* | 246 |
d-Ribose* | 35 |
d-Glucose* | 146 |
d-Mannose* | 48 |
d-Galactose* | 0 |
Cellobiose* | 71 |
Reduction reactionc | |
Pyruvaldehyde | 100 |
Dimethylglyoxal | 270 |
Glyoxylic acid | 36 |
Acetone | 0 |
Cyclopentanone | 0 |
Cyclohexanone | 4 |
3-Methyl-2-pentanone* | 13 |
d-Arabinose* | 0 |
d-Xylose* | 0 |
d-Glucose* | 0 |
Cellobiose* | 0 |
TABLE 2.
Apparent Km and Vmax values for TsAdh319Coenzyme or substrate | Apparent Km (mM) | Vmax (U mg−1) | kcat (s−1) |
---|---|---|---|
NADPa | 0.022 ± 0.002 | 0.94 ± 0.02 | 0.45 ± 0.01 |
NADPHb | 0.020 ± 0.003 | 3.16 ± 0.11 | 1.51 ± 0.05 |
2-Propanol | 168 ± 29 | 1.10 ± 0.09 | 0.53 ± 0.04 |
d-Xylose | 54.4 ± 7.4 | 1.47 ± 0.09 | 0.70 ± 0.04 |
Pyruvaldehyde | 17.75 ± 3.38 | 4.26 ± 0.40 | 2.04 ± 0.19 |
TABLE 3.
Effect of various ions and EDTA on TsAdh319aCompound | Concn (mM) | Relative activity (%) |
---|---|---|
None | 0 | 100 |
NaCl | 400 | 206 |
600 | 227 | |
4,000 | 230 | |
KCl | 600 | 147 |
2,000 | 200 | |
3,000 | 194 | |
MgCl2 | 10 | 78 |
CoCl2 | 10 | 105 |
NiSO4 | 10 | 100 |
ZnSO4 | 10 | 79 |
FeSO4 | 10 | 74 |
EDTA | 1 | 100 |
5 | 80 |
TABLE 4.
Influence of various solvents on TsAdh319 activityaSolvent | Relative activity (%)b | Relative activity (%)c | |
---|---|---|---|
Buffer without NaCl | Buffer with 600 mM NaCl | ||
None | 100 | 100 | 100 |
DMSOd | 98 | 0 | 40 |
DMFAe | 101 | 13 | 41 |
Methanol | 98 | 25 | 9 |
Acetonitrile | 95 | 0 | 0 |
Ethyl acetate | 47 | 0* | 33* |
Chloroform | 105 | 79* | 81* |
n-Hexane | 105 | 60* | 118* |
n-Decane | 36 | 91* | 107* |
18.
19.
Chris A. Whitehouse Carson Baldwin Rangarajan Sampath Lawrence B. Blyn Rachael Melton Feng Li Thomas A. Hall Vanessa Harpin Heather Matthews Marina Tediashvili Ekaterina Jaiani Tamar Kokashvili Nino Janelidze Christopher Grim Rita R. Colwell Anwar Huq 《Applied and environmental microbiology》2010,76(6):1996-2001
20.
Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression.Though Escherichia coli retains popularity as a host for recombinant protein production, it has technical limitations for expressing eukaryotic proteins. Host/protein incompatibility is critical, since eukaryotic proteins heterologously expressed in E. coli are recognized as a foreign object that can induce heat shock responses and drive protease-mediated degradation. E. coli contains several proteases situated in various intracellular compartments, including the cytoplasm, periplasm, inner membrane, and outer membrane (7, 9). While these proteases play a pivotal role in maintaining cell physiology by clearing up abnormal proteins (4), they can potentially degrade recombinant proteins (6). Nevertheless, the mechanisms associated with protein degradation and protein substrate specificity are incompletely understood.Since the proteolysis of recombinant proteins is a common issue, approaches to overcome this limitation have been extensively explored. These include using protease-deficient mutants as an expression host (10, 18), lowering the cultivation temperature (3), coexpressing folding factors (19, 20), applying protein fusion technology (2, 8), eliminating protease cleavage sites (5, 15), and modifying target protein hydrophobicity (11). While genetic elimination of proteolytically sensitive sequences without impacting the target protein''s bioactivity appears to be technically attractive, there is no effective prediction or identification of potential protease cleavage sites. Proteolytically sensitive sequences might not be specific to certain proteases. Various factors affecting the folding, solubility, steric hindrance, and binding of the substrate protein, e.g., neighboring domains or amino acids, can contribute to the sensitivity of proteolytic sites, making sequence manipulation for proteolysis alleviation more difficult.Herein, the recombinant protein''s sensitivity toward intracellular proteolysis was genetically alleviated to enhance the functional expression of PalB, an industrial enzyme with justified applications (1), in E. coli and to understand the relationship between proteolytic specificity and substrate protein sequence. Since there are three intramolecular disulfide bonds whose formation can potentially affect PalB structure and bioactivity, PalB expression in the oxidative periplasm of E. coli was explored (20, 22). However, the expressed PalB protein was extremely sensitive to intracellular proteolysis, resulting in complete degradation. To overcome this problem, PalB mutant variants that were stable against proteolysis were derived (Table (Table11).
Open in a separate windowaFor oligonucleotides, italic indicates mutated nucleotides, underlining indicates restriction site (silent mutation), and bold indicates a mutation codon.bSDM, site-directed mutagenesis.The results for functional expression of various PalB variants are summarized in Table Table22 and Fig. Fig.1.1. Neither PalB activity nor PalB-related polypeptide was detectable for the BL21(DE3)(pETG) culture sample, suggesting that the expressed gene product was degraded. Error-prone PCR (ep-PCR) was conducted using pETG as the template and LB-P10/LB-P11 as the primers. Potential mutants were screened on tributyrin agar plates. A colony developed a large halo, and the harboring plasmid (i.e., pETGM-2) was purified for sequencing. Two mutations, Leu149Val and Val223Ile, were identified in the expressed PalB mutant (i.e., M-2). High PalB activity was obtained for the BL21(DE3) (pETGM-2) culture sample. However, a PalB-related polypeptide was detected in both the soluble and insoluble fractions, implying potential protein misfolding. It was previously reported that the functional expression of PalB could be limited by disulfide bond formation associated with folding (21). It was intriguing to observe the structural effect of these two simple mutations on improving PalB stabilization and functional expression.Open in a separate windowFIG. 1.Western blotting analysis of the culture samples for the expression of various PalB mutants. Both soluble and insoluble fractions are shown. The number (n = 2 to 12) represents the PalB mutant (M-n) with the use of BL21(DE3) harboring the expression plasmid pETGM-n, summarized in Tables Tables11 and and2.2. “G” represents the control experiment using BL21(DE3) harboring pETG. “C” and “I” represent the cultures without and with IPTG induction, respectively.
Open in a separate windowaBL21(DE3) was used as an expression host to harbor various plasmids. PalB enzyme assay was conducted at 37°C and pH 8.0 using a pH stat with olive oil as a substrate. One unit of enzyme activity is defined as the amount of enzyme required to liberate one μmole of fatty acid per min.bOD600, optical density at 600 nm; w/o and w/IPTG, without and with isopropyl-β-d-thiogalactopyranoside, respectively.cThese activities represent IPTG-induced cultures. No activity was detected for all control cultures without IPTG induction. ND, not detected.Site-directed mutagenesis was conducted to introduce various single mutations of Leu149 and Val223 for a further understanding of the structural effect. Note that only hydrophobic amino acid residues were selected for the replacement to avoid any major structural disturbances. Among the five single mutations on Leu149, only Leu149Val and Leu149Ile were associated with a slight improvement in functional expression, and Leu149Val was more effective than Leu149Ile, whereas the others behaved similarly to the wild type. Among the five single mutations at Val223, only Val223Phe, Val223Gly, and Val223Ile showed a slight improvement in functional expression, with Val223Ile as the most effective one, whereas the others showed no improvement. The results suggest that Leu149 and Val223 are two amino acids critically affecting the proteolytic susceptibility of PalB in E. coli, with a synergistic effect on PalB stabilization from Leu149Val and Val223Ile mutations.Several amino acids were identified as being involved in the catalytic mechanism (17), such as Ser107-Asp189-His226, forming the catalytic triad, and Thr42 and Gly108, acting as the hydrogen bond donors in the oxyanion hole of the active site. However, Leu149 is located at the entrance of the partially formed lid of the protein structure, whereas Val223 is situated near one of the catalytic triad residues, His226, in the active site. The substitutions at Leu149 and/or Val223 with a PalB stabilization effect potentially convert the overall protein conformation into a less proteolytically sensitive form without changing the enzyme kinetics or activity. It was interesting to observe a similarly positive effect upon replacing Val223 with either a bulky Phe or a small Gly, implying that the molecular size of this amino acid residue was not critical for alleviation of the proteolytic sensitivity. Note that PalB-related polypeptide was present in the insoluble fraction for all the PalB mutants with improved functional expression, suggesting another expression hurdle of protein misfolding.To overcome the protein misfolding, coexpression of two periplasmic folding factors, DsbA and DsbC, was explored, and the cultivation results are summarized in Fig. Fig.2.2. DsbA coexpression significantly boosted functional expression, with specific PalB activity of more than twofold that of the control culture and a slight reduction of insoluble PalB. However, the improving effect associated with DsbC coexpression was minimal. The results suggest that the initiation but not isomerization for disulfide bond formation in the periplasm became limiting. The chaperone activity associated with DsbA could assist the folding of the expressed PalB double mutant.Open in a separate windowFIG. 2.Effect of DsbA or DsbC coexpression on the functional expression of the PalB double mutant. Cultivations were performed in a bioreactor containing 1 liter LB medium and operated at pH 7.0, 28°C, and 650 rpm. The cultures were induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside and/or 0.2 g/liter arabinose at a ∼0.5 optical density at 600 nm (OD600) cell density. (A) Time profiles of cell density; (B) time profiles of the specific PalB activity [note that there was no detectable activity for BL21(DE3) (pETG) culture samples]; (C) Western blotting analysis of the final samples of the four cultures. Both soluble and insoluble fractions are shown. Lanes 1 and 5, BL21(DE3) (pETG); lanes 2 and 6, BL21(DE3) (pETGM-2); lane 3 and 7, BL21(DE3) (pETGM-2, pARDsbC); lanes 4 and 8, BL21(DE3) (pETGM-2, pARDsbA).While many studies based on rational mutagenesis, directed evolution, and gene shuffling are conducted to derive PalB mutants with improved enzyme properties, such as thermostability, bioactivity, or enantioselectivity (12-14, 16), this is an approach to improve protein manufacturing by deriving the variants with less proteolytic susceptibility. Though it has been perceived that the proteolytic specificity can be intrinsically determined by the protein substrate sequence, experimental demonstration of this is rarely reported. This study complements the lack of such experimental demonstration by showing the proteolytic specificity and sensitivity of PalB heterologously expressed in E. coli can be drastically altered by simple amino acid substitutions. It also demonstrates the application of molecular manipulation to enhance recombinant protein production in E. coli. 相似文献
TABLE 1.
Strains, plasmids, and oligonucleotidesStrain, plasmid, or oligonucleotide | Relevant genotype or DNA sequencea |
---|---|
Strain | |
BL21(DE3) | F−ompT dcm lon hsdSB (rB− mB−) gal λ(DE3[lacIind1sam7 nin5 lacUV5-T7 gene 1]) |
Plasmids | |
pARDsbA (20) | ParaB::dsbA, Ori (pACYC184), Cmr |
pARDsbC (20) | ParaB::dsbA, Ori (pACYC184), Cmr |
pETG (20) | PT7-pelB::palB, Ori (pBR322), Apr |
pETGM-2 | pETG derivative containing two mutations of Leu149Val and Val223Ile in palB (by ep-PCR with pETG and LB-P10/LB-P11) |
pETGM-3 | pETG derivative containing mutation of Leu149Gly in palB (by SDMb with pETG and P104/P114) |
pETGM-4 | pETG derivative containing mutation of Leu149Val in palB (by SDM with pETG and P103/P113) |
pETGM-5 | pETG derivative containing mutation of Leu149Ala in palB (by SDM with pETG and P105/P115) |
pETGM-6 | pETG derivative containing mutation of Leu149Met in palB (by SDM with pETG and P106/P116) |
pETGM-7 | pETG derivative containing mutation of Leu149Ile in palB (by SDM with pETG and P107/P117) |
pETGM-8 | pETG derivative containing mutation of Val223Phe in palB (by SDM with pETG and P112/P122) |
pETGM-9 | pETG derivative containing mutation of Val223Met in palB (by SDM with pETG and P111/P121) |
pETGM-10 | pETG derivative containing mutation of Val223Ala in palB (by SDM with pETG and P110/P120) |
pETGM-11 | pETG derivative containing mutation of Val223Gly in palB (by SDM with pETG and P109/P119) |
pETGM-12 | pETG derivative containing mutation of Val223Ile in palB (by SDM with pETGM-2 and P136/P137, i.e., by reverting the mutation of Val149 to Leu in pETGM-2) |
Oligonucleotides | |
LB-P10 | 5′-GGCCATGGGTCTACCTTCCGGTTCGG-3′ |
LB-P11 | 5′-CTGAATTCTCAGGGGGTGACGATGCCGGAGCAGG-3′ |
P103 | 5′-CCTCTCGATGCAGTCGCGGTTAGTGCACCC-3′ |
P113 | 5′-GGGTGCACTAACCGCGACTGCATCGAGAGG-3′ |
P104 | 5′-CCTCTCGATGCAGGCGCCGTTAGTGCACCC-3′ |
P114 | 5′-GGGTGCACTAACGGCGCCTGCATCGAGAGG-3′ |
P105 | 5′-CCTCTCGATGCAGCCGCGGTTAGTGCACCC-3′ |
P115 | 5′-GGGTGCACTAACCGCGGCTGCATCGAGAGG-3′ |
P106 | 5′-CCTCTCGATGCCATGGCGGTTAGTGCACCC-3′ |
P116 | 5′-GGGTGCACTAACCGCCATGGCATCGAGAGG-3′ |
P107 | 5′-CCTCTCGATGCGATCGCGGTTAGTGCACCC-3′ |
P117 | 5′-GGGTGCACTAACCGCGATCGCATCGAGAGG-3′ |
P109 | 5′-GGGCCGCTGTTCGGGATCGACCATGCAGGC-3′ |
P119 | 5′-GCCTGCATGGTCGATCCCGAACAGCGGCCC-3′ |
P110 | 5′-GGGCCGCTGTTCGCGATCGACCATGCAGGC-3′ |
P120 | 5′-GCCTGCATGGTCGATCGCGAACAGCGGCCC-3′ |
P111 | 5′-GGGCCGCTGTTCATGATCGACCATGCAGGC-3′ |
P121 | 5′-GCCTGCATGGTCGATCATGAACAGCGGCCC-3′ |
P112 | 5′-GGGCCGCTGTTCTTTATCGACCATGCAGGC-3′ |
P122 | 5′-GCCTGCATGGTCGATAAAGAACAGCGGCCC-3′ |
P136 | 5′-CCTCTCGATGCACTGGCGGTTAGTGCACCC-3′ |
P137 | 5′-GGGTGCACTAACCGCCAGTGCATCGAGAGG-3′ |
TABLE 2.
Cultivation performance for production of PalB mutant variantsaPlasmid | Cell density (OD600)b
| Activity (U/liter/OD600)c | Mutation in PalB | |
---|---|---|---|---|
w/o IPTG | w/IPTG | |||
pETG | 3.1 ± 0.0 | 3.3 ± 0.1 | ND | None |
pETGM-2 | 3.4 ± 0.1 | 3.3 ± 0.0 | 155 | Leu149Val, Val223Ile |
pETGM-3 | 3.2 ± 0.1 | 3.3 ± 0.0 | ND | Leu149Gly |
pETGM-4 | 3.4 ± 0.1 | 3.6 ± 0.1 | 60 | Leu149Val |
pETGM-5 | 3.3 ± 0.1 | 3.5 ± 0.1 | ND | Leu149Ala |
pETGM-6 | 2.7 ± 0.1 | 3.0 ± 0.1 | ND | Leu149Met |
pETGM-7 | 3.5 ± 0.0 | 3.2 ± 0.0 | 38 | Leu149Ile |
pETGM-8 | 3.0 ± 0.0 | 3.6 ± 0.0 | 50 | Val223Phe |
pETGM-9 | 2.9 ± 0.1 | 2.8 ± 0.0 | ND | Val223Met |
pETGM-10 | 3.4 ± 0.1 | 3.3 ± 0.1 | ND | Val223Ala |
pETGM-11 | 3.5 ± 0.0 | 3.5 ± 0.1 | 30 | Val223Gly |
pETGM-12 | 3.6 ± 0.1 | 3.5 ± 0.1 | 57 | Val223Ile |