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1.
Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases
(DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids
with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic
d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His 6-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg −1 protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His 6-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co 2+ and Mn 2+ ions. His 6-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies ( kcat/ Km) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s −1 mM −1, respectively. 相似文献
2.
The xylose reductase (XR) gene ( xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His 6-tagged fusion protein. The recombinant XR had K m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was
specific for d-xylese. 相似文献
3.
The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession
no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized
sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP)
can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His 6-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins
in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts
from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP
were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added
to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate
that ER-sHSP can function as a molecular chaperone in vitro.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid
ajmaline. The enzyme was cloned by a “reverse-genetic” approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His 6-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl
function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids.
The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the
aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed
mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds
of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of
plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
The nucleotide sequences reported in this article have been submitted to the Gene Bank under Accession No: AY766462. 相似文献
5.
In the present study, the biophysical properties of His 6-tagged Bacillus stearothermophilus aminopeptidase II (His 6-tagged BsAmpII) are characterized in detail by gel-filtration, analytical ultracentrifugation, and various spectroscopic techniques. Using size-exclusion chromatography and analytical ultracentrifugation, we demonstrate that His 6-tagged BsAmpII exists predominantly as a dimer in solution. The enzyme is active and stable at pHs ranging from 6.5 to 8.5. Far-UV circular dichroism analysis reveals that the secondary structures of His 6-tagged BsAmpII are significantly altered in the presence of SDS, whereas the presence of 5–10% acetone and ethanol was harmless to the folding of the enzyme. Thermal unfolding of His 6-tagged BsAmpII was found to be irreversible and led to the formation of aggregates. The native enzyme started to unfold beyond 0.6 M guanidine hydrochloride and had a midpoint of denaturation at 1.34 M. This protein remained active at concentrations of urea below 2.7 M but experienced an irreversible unfolding by >5 M denaturant. Taken together, this work lays a foundation for potential biotechnological applications of His 6-tagged BsAmpII. 相似文献
6.
We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His 6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His 6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His 6 tag is removed by His 6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His 6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification. 相似文献
7.
The Arabidopsis thaliana mutant psbo1 has recently been described and characterized. Loss of expression of the PsbO-1 protein leads to a variety of functional perturbations including elevated levels of the PsbO-2 protein and defects on both the oxidizing- and reducing-sides of Photosystem II. In this communication, two plant lines were produced using the psbo1 mutant as transgenic host, which contained an N-terminally histidine 6-tagged PsbO-1 protein. This protein was expressed and correctly targeted into the thylakoid lumen. Immunological analysis indicated that different levels of expression of the modified PsbO-1 protein were obtained in different transgenic plant lines and that the level of expression in each line was stable over several generations. Examination of the Photosystem II closure kinetics demonstrated that the defective double reduction of Q B and the delayed exchange of Q BH 2 with the plastoquinone pool which were observed during the characterization of the psbo1 mutant were effectively restored to wild-type levels by the His 6-tagged PsbO-1 protein. Flash fluorescence induction and decay were also examined. Our results indicated that high expression of the modified PsbO-1 was required to increase the ratio of PS II α/PS II β reaction centers to wild-type levels. Fluorescence decay kinetics in the absence of DCMU indicated that the expression of the His 6-tagged PsbO-1 protein restored efficient electron transfer to Q B, while in the presence of DCMU, charge recombination between Q A− and the S 2 state of the oxygen-evolving complex occurred at near wild-type rates. Our results indicate that high expression of the His 6-tagged PsbO-1 protein efficiently complements nearly all of the photochemical defects observed in the psbo1 mutant. Additionally, this study establishes a platform on which the in vivo consequences of site-directed mutagenesis of the PsbO-1 protein can be examined. 相似文献
8.
To express Escherichia coli novablue dipeptidyl carboxypeptidase ( EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE- EcDCP. His 6-tagged EcDCP (His 6- EcDCP) was over-expressed in E. coli M15 (pQE- EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity
by Ni 2+-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His 6- EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His 6- EcDCP was stimulated by 1.5 fold. The K
M and k
cat values of the enzyme for N-benzoyl- l-glycyl- l-histidyl- l-leucine were 1.83 mM and 168.3 s −1, respectively. His 6- EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected
by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His 6- EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17%
α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme. 相似文献
9.
Cobalt-chelated magnetic (Fe 3O 4-AA-ANTA-Co 2 +) particles were prepared and one-step purification and immobilization of His 6-tagged Escherichia coli γ-glutamyltranspeptidase (His 6- EcGGT) using these particles were evaluated. The optimal conditions for the adsorption of His 6- EcGGT to Fe 3O 4-AA-ANTA-Co 2 + particles were found to be 24.7 U g –1 adsorbent, pH 6.5, 300 mM NaCl and 30 min incubation at 4°C, while the elution solution was optimized to be 50 mM phosphate buffer (pH 6.5) containing 150 mM imidazole and 300 mM NaCl. The immobilized His 6- EcGGT was recycled five times without significant loss of GGT activity. The average yield rate for the synthesis l-theanine from glutamine and ethylamine reached 56.7%. These results indicate that one-step affinity purification and immobilization of His 6- EcGGT by Fe 3O 4-AA-ANTA-Co 2 + particles might serve as an effective process for industrial application. 相似文献
10.
A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His 6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 m M IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K
m and V
max values for γ-glutamyl- p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μ M and 53.7 × 10 −3 m M min −1, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 m M
L-Gln, 40 m M ethylamine, and 1.04 U His 6-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained. 相似文献
12.
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 (His 6-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His 6-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 His 6-tag (designated His 6t-S-CD) was secreted as a monomer. His 6t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His 6t-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. 相似文献
13.
Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His 6- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive
bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin
amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene
sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant
proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins.
After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His 6-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified
levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru.
R. Biedendieck and R. Beine contributed equally to this work. 相似文献
14.
Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His 6-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind β-lactams, indicating these putative PBPs have penicillin-binding activity. 相似文献
15.
The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX 1 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase
was expressed in Pichia Pastoris X 33 and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA
resin). High level expression of the lipase by Pichia Pastoris X 33 cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C;
the specific activity of the purified His 6-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of
Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His 6-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H
was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His 6-ROLw) and the mutant (His 6-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have
concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H)
could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface
and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among
Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis. 相似文献
16.
Summary Histidine-436 of a truncated Bacillus sp. strain TS-23 α-amylase (His 6-tagged ΔNC) has been known to be responsible for thermostability of the enzyme. To understand further the structural role of this residue, site-directed mutagenesis was conducted to replace His-436 of His 6-tagged ΔNC with aspartate, lysine, tyrosine or threonine. Starch-plate assay showed that all Escherichia coli M15 transformants conferring the mutated amylase genes retained the amylolytic activity. The over-expressed proteins have been purified to near homogeneity by nickel-chelate chromatography and the molecular mass of the purified enzymes was approximately 54 kDa. The specific activity for H436T was decreased by more than 56%, while H436D, H436K, and H436Y showed a higher activity to that of the wild-type enzyme. Although the mutations did not lead to a significant change in the Km value, more than 66% increase in the value of catalytic efficiency ( kcat/ Km) was observed in H436D, H436K, and H436Y. At 70 °C, H436D exhibited an increased half-life with respect to the wild-type enzyme. 相似文献
17.
Lipoprotein-associated phospholipase A 2 (Lp-PLA 2) has been shown to play a crucial role in atherosclerosis, and has been proposed as a promising target for drug discovery.
Here, we cloned the Lp-PLA
2 gene from differentiated THP-1 cells, and inserted a carboxy-terminal His 6-tagged version of the gene into the pPIC9 Pichia expression vector. The Lp-PLA 2 fusion protein was successfully expressed in Pichia pastoris expression system and could be rapidly purified to apparent homogeneity using a single-step purification method. The activity
of our recombinant Lp-PLA 2 was strong when [ 3H] PAF was used as a substrate, and the Lp-PLA 2 inhibitor SB435495 exhibited an inhibitory curve against the recombinant Lp-PLA 2 (IC 50=15.93±1 μ M). This novel recombinant Lp-PLA 2 could prove useful as a screening model for Lp-PLA 2 inhibitors, and may facilitate further investigation of this protein in atherosclerosis. 相似文献
18.
The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His6)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His6-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His6-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling. 相似文献
19.
For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32
to generate pQE-LAPII. The His 6-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity
of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5
kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60°C and 8.0, respectively. Under optimal condition,
the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His 6-tagged enzyme was stimulated by Co 2+ ions, but was strongly inhibited by Cu 2+ and Hg 2+ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co 2+ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that
the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.
Received: 16 August 2002 / Accepted: 4 September 2002 相似文献
20.
Poly- N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion
to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures
and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling
onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1
and β1,3- N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture
of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model
galectin His 6CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated
binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized
surfaces by imitating the natural ECM microenvironment.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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