Overexpression,Purification, and Characterization of the Recombinant Leucine Aminopeptidase II of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>

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 His₆-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 His₆-tagged enzyme was stimulated by Co²⁺ ions, but was strongly inhibited by Cu²⁺ and Hg²⁺ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co²⁺ 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     

Cloning,expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.     

Type II isopentenyl diphosphate isomerase from Synechocystis sp. strain PCC 6803

Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His(6)-tagged protein was produced in Escherichia coli and purified by Ni(2+) chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg(2+) for activity; K(m)(IPP) was 52 microM, and k(cat)(IPP) was 0.23 s(-1).     

Expression,purification, and characterization of human tyrosyl-tRNA synthetase

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.     

Overexpression of a recombinant gamma-glutamyltranspeptidase from Escherichia coli Novablue

A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene, lacking the first 48-bp coding sequence for part of the signal sequence, was amplified by polymerase chain reaction (PCR) and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees 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 determined to be 41 and 21 kDa respectively by SDS-PAGE, indicating the precursor EcGGT still undergoes the post-translational processing even in the truncation of signal sequence. His6-tagged EcGGT migrated relative to the molecular mass of approximately 120 kDa and its heterodimeric structure was confirmed by a native-PAGE gel.     

Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells.

p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells. In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15. The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity. From the poly(A)(+) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His(6)-tagged recombinant p15 was produced in Escherichia coli. The recombinant protein purified by a single step on Ni(2+) agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay. The effect of His(6)-p15 was blocked by nicardipine, suggesting that Ca(2+) influx through the L-type Ca(2+) channels is essential for its neurite-inducing activity. In addition, mutational analysis of His(6)-p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity.     

High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.     

Soluble expression in Escherichia coli,purification and characterization of a human TF-1 cell apoptosis-related protein TFAR19

A novel human TF-1 cell apoptosis-related protein, TFAR19, cloned from a human leukemia cell line, TF-1, was first overexpressed in Escherichia coli with the sequence Met-Gly-His(6)-Gly-Thr-Asn-Gly, a hexahistidine sequence followed by a hydroxylamine cleavage site attached to its amino terminus. The resulting protein was soluble and single-step purified to homogeneity by metal chelating affinity chromatography. After cleavage of the purified His(6)-tagged TFAR19 sample with hydroxylamine, highly purified untagged TFAR19 protein was then obtained through an FPLC Resource Q column. The structural characteristics and function of the His(6)-tagged and untagged TFAR19 proteins were studied using circular dichroism, intrinsic fluorescence, and ANS-binding fluorescence spectra and apoptosis activity assay. The results show that alpha-helix is the main secondary structure of the proteins and the two forms of TFAR19 protein fold properly, which correspond well to their apoptosis activity expression. The results also indicate that the extra sequence including the His(6)-tag fused to the N-terminus of TFAR19 protein has a minimal effect on its structure and function, suggesting that the His(6)-tagged TFAR19 protein could be further used as an immobilized target for finding potential proteins which interact with TFAR19 from a cDNA library using in vitro ribosome display technique.     

A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel protocol for producing recombinant Moloney murine leukemia virus (MMLV-RT) in Escherichia coli is reported. The optimized coding sequence for mature MMLV-RT was cloned into pET28a and over-expressed as an N-terminal His6-tagged fusion protein. An enterokinase (EK) recognition site was introduced between the His6-tag and MMLV-RT to release tag-free enzyme. Optimal expression of soluble His6-MMLV-RT was achieved by chaperone co-expression and lower temperature fermentation. The His6-tagged enzyme was first purified by Ni²⁺ affinity chromatography. The bound enzyme was then eluted by EK digestion and the eluate was purified on an anion-exchange Q column to remove DNA and EK. Twenty-one milligram MMLV-RT was obtained from 1 l of bacterial culture.     

An improved SUMO fusion protein system for effective production of native proteins

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.     

Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis

A nitrilase-like protein from Arabidopsis thaliana (NLP1) was expressed in Escherichia coli as a His(6)-tagged protein and purified to apparent homogeneity by Ni(2+)-chelate affinity chromatography. The purified enzyme showed N-carbamoylputrescine amidohydrolase activity, an enzyme involved in the biosynthesis of polyamines in plants and bacteria. N-carbamoylputrescine amidohydrolase activity was confirmed by identification of two of the three occurring products, namely putrescine and ammonia. In contrast, no enzymatic activity could be detected when applying various compounds including nitriles, amines, and amides as well as other N-carbamoyl compounds, indicating the specificity of the enzyme for N-carbamoylputrescine. Like the homologous beta-alanine synthases, NLP1 showed positive cooperativity toward its substrate. The native enzyme had a molecular mass of 279 kDa as shown by blue-native polyacrylamide gel electrophoresis, indicating a complex of eight monomers. Expression of the NLP1 gene was found in all organs investigated, but it was not induced upon osmotic stress, which is known to induce biosynthesis of putrescine. This is the first report of cloning and expression of a plant N-carbamoylputrescine amidohydrolase and the first time that N-carbamoylputrescine amidohydrolase activity of a recombinant protein could be shown in vitro. NLP1 is one of the two missing links in the arginine decarboxylase pathway of putrescine biosynthesis in higher plants.     

Purification,characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.     

Cloning, expression, and purification of the His(6)-tagged thermostable beta-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme

In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid. The nucleotide sequence revealed that beta-galactosidase of P. woesei consists of 510 amino acids and has a molecular weight of 59, 056 kDa (GenBank Accession No. AF043283). It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S. D $$;abrowski, J. Maciuńska, and J. Synowiecki, 1998, Mol. Biotechnol. 10, 217-222). In this study we presented a new expression system for producing P. woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase). The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. The enzyme was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni(2+)-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability. This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase.     

An efficient method for the overexpression and purification of active tyrosinase from Streptomyces castaneoglobisporus

The melanin-synthesizing gene operon cloned from Streptomyces castaneoglobisporus HUT6202 consists of two genes, designated tyrC and orf378, which encode apotyrosinase (TYRC) and its activator protein (ORF378), respectively. We have suggested that ORF378 may facilitate the incorporation of Cu(II) into apotyrosinase to express tyrosinase activity. To overproduce ORF378 and TYRC in Escherichia coli BL21(DE3)-pLysS, tyrC, and orf378 were independently but not polycistronically placed under the control of a T7 promoter in a vector, pET-21a(+). His(6)-tagged TYRC and His(6)-tagged ORF378 were simultaneously overproduced in an E. coli strain harboring a plasmid, designated pET-mel2, and the two proteins were co-purified with a Ni(II)-bound affinity column. Gel filtration analysis revealed that the two proteins form a heterodimer complex. The complexed protein was retrieved at a high efficiency (11 mg/L). To obtain an active TYRC, which is a Cu(II)-bound form of tyrosinase, we constructed pET-mel3 that carries orf378 without His(6)-tag and His(6)-tagged tyrC. After the cell-free extract from E. coli harboring pET-mel3 was subjected to Cu(II)-bound affinity column chromatography, His(6)-tagged TYRC, eluted from the column, exhibited the tyrosinase activity. The k(cat) and K(m) values for l-3,4-dihydroxyphenylalanine (l-DOPA) of His(6)-tagged TYRC, which catalyzes the oxidation of l-DOPA to dopaquinone, were 880+/-80s(-1) and 8.1+/-0.9 mM, respectively.     

Expression,oxidative refolding,and characterization of six-histidine-tagged recombinant human LECT2, a 16-kDa chemotactic protein with three disulfide bonds

Human LECT2 is a 16-kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulfide bonds. Here, we present the oxidative refolding of (His)(6)-LECT2, an N-terminally (His)(6)-tagged recombinant protein of human LECT2. (His)(6)-LECT2 was overproduced in Escherichia coli in the form of insoluble aggregates, solubilized with 8 M urea in the presence of 10 mM DTT, and purified and refolded on Ni-NTA agarose by lowering the urea concentration before the elution. This process, however, gave a mixture of oligomers of (His)(6)-LECT2 as well as the monomer, whose composition was as low as 36%. The oligomers formed as a result of incorrect intermolecular disulfide bonds. After the refolding on Ni-NTA agarose (step 1), the disulfide bonds were shuffled using a glutathione redox buffer (step 2) and the remaining thiols were completely oxidized (step 3) to improve the yield of correctly folded, monomeric (His)(6)-LECT2. The monomer composition was significantly improved to 81% by the three-step refolding method and the monomer thus obtained was shown to have the same conformation as the authentic LECT2 produced in CHO cells by CD and NMR spectroscopies. The yield of (His)(6)-LECT2 was 1.0 mg/L E. coli culture and was 16 times as high as that in our previous report, in which (His)(6)-LECT2 was purified from the soluble fractions of E. coli cell lysates.     

Cloning, expression, purification, and characterization of a novel epoxide hydrolase from Aspergillus niger SQ-6

A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.     

An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.     

Heterologous expression, purification, and characterization of a highly active xylose reductase from Neurospora crassa

A xylose reductase (XR) gene was identified from the Neurospora crassa whole-genome sequence, expressed heterologously in Escherichia coli, and purified as a His6-tagged fusion in high yield. This enzyme is one of the most active XRs thus far characterized and may be used for the in vitro production of xylitol.     

Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli

The SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6x His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54kDa. The optimal temperature and pH of the purified rSAP6 were 40 degrees C and 3.4, respectively. The enzyme was stable below 45 degrees C and between pH 2.6 and 5.0. The results show that Mn(2+) had an activating effect on the enzyme, while Cu(2+), Mg(2+), Zn(2+) and Ag(+) acted as inhibitors of the enzyme. However, Ca(2+) had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.     

Preparation of recombinant six-histidine-tagged human LECT2, a chemotactic protein to neutrophils, in Escherichia coli

LECT2 is a chemotactic protein to neutrophils. A recombinant six-histidine-tagged human LECT2, (His)₆-LECT2, was expressed in E. coli using a pET21a(+) vector. The (His)₆-LECT2 was purified from the soluble fraction in E. coli as a single band in sodium dodesyl sulfate/polyacrylamide gel electrophoresis using three steps of column chromatography with Ni²⁺-charged nitrilo-triacetic acid (Ni-NTA) agarose, DEAE-Sepharose, and CM-Sepharose. The purified (His)₆-LECT2 was yielded with 96 μg from the soluble fraction of 1,500 ml culture of E. coli. The circular dichroism spectrum of (His)₆-LECT2 showed the folded structure, which is rich in β-sheet structure and rare in α-helix. This revised version was published online in June 2006 with corrections to the Cover Date.