A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A₂ ([Lys⁴⁹]PLA₂), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.     

Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, <Emphasis Type="Italic">Alkalimonas collagenimarina</Emphasis> AC40<Superscript>T</Superscript>

The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40^T, was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0–9.5 and 45°C in 100 mM glycine–NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40^T was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.     

Cloning,Characterization, and Expression of the Gene Encoding Alkaline Protease in the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> 10

The alkaline protease structural gene (ALP1 gene) was isolated from both the genomic DNA and cDNA of Aureobasidium pullulans 10 by inverse PCR and RT-PCR. An open reading frame of 1248 bp encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. The gene contained two introns, which had 54 bp and 50 bp, respectively. The promoter of ALP1 gene was located from -62 to -112 and had two CCAAT boxes and one TATA box. The terminator of ALP1gene contained the sequence with a hairpin structure (AAAAAGTT TGGTTTTT). The protein sequence deduced from ALP1 gene exhibited 55.24%, 50.35%, and 31.68% identity with alkaline proteases from Aspergillus fumigatus, Acremonium chrysogenum, and Yarrowia lipolytica, respectively. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2% casein and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP1 gene is expressed in Y. lipolytica and the expressed alkaline protease is secreted into the medium.     

An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: gene cloning, expression and characterization

A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Cloning of Transglutaminase Gene from Streptomyces fradiae and its Enhanced Expression in the Original Strain

The transglutaminase (TGase) gene of Streptomyces fradiae was cloned. It had an ORF of 1242 bp, encoding a presumed prepro-region of 82 amino acids and a mature TGase of 331 amino acids. Enhanced expression of the TGase was achieved by introducing another copy of TGase gene into the original host genome which was driven by the strong constitutive promoter, “ermE up”, and shown to be expressed at the mRNA and protein levels. TGase activity in the recombinant strain (3.2 U/ml) was improved 1.3-fold when compared to that normally expressed in the original strain (2.4 U/ml). The specific enzyme activity in the recombinant strain (3.8 U/mg) was double that of the original strain (1.9 U/mg).     

Immunological Detection of Serpin in the Fall Webworm, Hyphantria cunea and Its Inhibitory Activity on the Prophenoloxidase System

We previously identified a serine type protease inhibitor (serpin) cDNA, using PCR-based differential display, in the fall webworm which was up-regulated following a bacterial challenge (Shin et al., 1998). The serpin cDNA was inserted into an expression vector and the serpin protein was expressed in Escherichia coli. In order to investigate the action of serpin in vivo, we examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabbit injected with recombinant serpin. H. cunea serpin was found mainly in the plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis. The concentration of serpin in the plasma was slightly increased following bacterial challenge. A new 50.5 kDa (approx.) band was detected post E. coli and distilled water injection. Both E. coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma, although E. coli injection produced a larger increase in activity. Hyphantria serpin probably participates in negative regulation of the prophenoloxidase (proPO) cascade. Recombinant serpin inhibits PO activity in the hemocyte lysate fraction activated by LPS. There is a similarity between the P₂–P₂ region (NKFG) of the serpin reactive site loop and the S₂–S₂ region (NRFG) of the insect proPO maturation site. This indicates a form of competitive inhibition of serpin against a protease involved in the activation of proPO. A tyrosine residue in the P₁₁ region of serpin, which is conserved in the S₁₁ regions of all known proPOs maturation sites, provides further support for this hypothesis.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Dimethylsulfoxide and sodium butyrate enhance the production of recombinant cyclooxygenase 2 in stably transformed Drosophila melanogaster S2 cells

Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni²⁺-affinity fractionation to a specific activity of 24 800 U mg^–1 protein. The peak level of recombinant Cox 2 production was 1.6 g (10⁷ cells)^–1, seven days after induction with 0.5 mM CuSO₄. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively.     

A new monocupin quercetinase of Streptomyces sp. FLA: identification and heterologous expression of the queD gene and activity of the recombinant enzyme towards different flavonols

The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus ^T, was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3′,4′-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis₆) was purified. Several flavonols were converted by QueDHis₆, whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.     

Neisseria gonorrhoeae IgA protease. Secretion and implications for pathogenesis

A cloned 5 bk DNA fragment from Neisseria gonorrhoeae strain MS11 promotes expression and excretion of IgA protease in E. coli and other Gram-negative hosts. DNA sequencing reveals a large open reading frame coding for a prcursor molecule of 169 kd. The 106 kd mature IgA protease is released from the bacteria in conjunction with a 15 kd soluble precursor segment, the -protein. In contrast, the carboxy terminal portion of the precursor, the -protein (45 kd), remains associated with the outer bacterial membrane. The three proteins result form autoproteolytic cleavage at sites in the precursor which are similar to the target site in IgA1. Consensus sequences of the specific cleavage sites are found in a number of relevant human proteins. IgA protease may therefore have other natural substrates besides IgA1. The soluble -protein as well as the membrane bound -protein, both associated with IgA protease, may confer additional virulence functions to the gonococcus.     

Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His₆-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His₆-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His₆-tag (designated His₆t-S-CD) was secreted as a monomer. His₆t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His₆t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.     

Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): Identification of catalytic and cellulose-binding domains

Summary The nucleotide sequence of the celZ gene coding for a thermostable endo--1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 by which encodes a preprotein of 986 amino acids with an M_r of 109000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fuorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.     

Hyperproduction of a recombinant fusion protein of Staphylococcus aureus V8 protease in Escherichia coli and its processing by OmpT protease to release an active V8 protease derivative

The expression of a recombinant fusion protein including Staphylococcus aureus V8 protease was studied by using Escherichia coli as the host strain. When the mature V8 protease was expressed as a fusion protein with a truncated E. coli \-galactosidase (\-gal97S4D), we could not obtain a sufficient amount of the enzyme because of the toxicity resulting from the expressed protease activity. Synthesis of V8 protease was increased by constructing a sandwich-type fusion protein consisting of \-gal197S4D, a V8 protease derivative with the 56 C-terminal amino acids deleted (V856) and a truncated aminoglycoside-3'-phosphotransferase. This fusion protein was successfully produced as inactive inclusion bodies. To release the V856 protease from the fusion protein, we developed a novel processing method using an endogeneous E. coli OmpT protease, which can recognize the dibasic amino acid residues located in the linker peptides of the fusion protein. After solubilizing the inclusion bodies with urea, the V856 protein was automatically released from the fusion protein by the OmpT protease, which was coprecipitated with the inclusion bodies. The V856 protease thus obtained showed the same enzymatic activity as that of the native V8 protease. We demonstrate in this study that the N-terminal prepro sequence and the C-terminal repeated sequence of this enzyme are not necessary for its enzymatic activity and protein folding.     

Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 -glycosidase (Tca -glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a M _r of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca -glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg^–1 protein.     

The production of soluble pyrroloquinoline quinone glucose dehydrogenase by Klebsiella pneumoniae, the alternative host of PQQ enzymes

Klebsiella pneumoniae, which produces PQQ and is available for use with a conventional expression vector system, was selected as the host strain for soluble PQQ glucose dehydrogenase (PQQGDH-B) production. The recombinant K. pneumoniaeexpressed PQQGDH-B in its holo-form at about 18000 U l^–1, equal to that achieved in recombinant Escherichia coli. The signal sequence of recombinant PQQGDH-B produced by K. pneumoniaewas correctly processed. K. pneumoniaecan become an alternative host microorganism not only for PQQGDH-B production but also for recombinant PQQ enzymes production.     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

Molecular cloning,recombinant gene expression,and antifungal activity of cystatin from taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> cv. Kaosiung no. 1)

A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5-/3-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST–CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 g recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150–200 g/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.     

Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain ofAzotobacter vinelandii

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N,N-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O₂-limited chemostat cultures. The results showed that the mutant strain grew and used O₂ less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochromeo,a ₁, oxidase branch of the respiratory chain may be important in the physiology ofA. vinelandii under O₂-limiting growth conditions.     

Alkaline Protease Gene Cloning from the Marine Yeast Aureobasidium pullulans HN2-3 and the Protease Surface Display on Yarrowia lipolytica for Bioactive Peptide Production

The alkaline protease genes (cDNAALP2 gene and ALP2 gene) were amplified from complementary DNA (cDNA) and genomic DNA of the marine yeast Aureobasidium pullulans HN2-3, respectively. An open reading frame of 1,248 bp encoding a 415-amino acid protein with a calculated molecular weight of 42.9 kDa was characterized. The ALP2 gene contained two introns, which had 54 and 52 bp, respectively. When the cDNAALP2 gene was cloned into the multiple cloning sites of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying protease could form a clear zone on the double plate containing milk protein and had protease activity. The cells displaying alkaline protease were also found to be able to produce bioactive peptides from different sources of proteins. The peptides produced from single-cell protein of marine yeast strain G7a had the highest angiotensin-converting enzyme inhibitory activity, while the peptides produced from spirulina protein had the highest antioxidant activity. This is the first report that the yeast cells displaying alkaline protease were used to produce bioactive peptides.