Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 μmol min(-1) mg(-1) protein.     

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.     

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.     

High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli

The hybrid antibacterial peptide CA-MA [cecropinA(1-8)-magainin2(1-12)] with potent antimicrobial properties but no hemolytic activity is a potential alternative antibiotic. To explore a new approach for high-level expression of the hybrid peptide CA-MA in Escherichia coli, the sequence of ubiquitin (UBI) from housefly was inserted into the plasmid pQE30 to construct the vector pQEUBI. The cDNA fragment encoding CA-MA with preferred codons of E. coli was obtained by recursive PCR (rPCR) and cloned into the vector pQEUBI to express the fusion protein (His)(6)-UBI-CA-MA. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 36% of the total proteins). With (His)(6)-tag, the fusion protein was easily purified by Ni-NTA chromatography and 36 mg of fusion protein was purified from 1L of culture medium. The fusion protein was efficiently cleaved by ubiquitin C-terminal hydrolase (UCH), yielding recombinant CA-MA with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant CA-MA was purified to homogeneity by reversed-phase HPLC and 6.8mg of pure active CA-MA was obtained from 1L culture medium. Analysis of recombinant CA-MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA-MA was 2559Da, which perfectly matches the mass (2559Da) calculated from the amino acid sequence. Analysis of CA-MA by circular dichroism (CD) revealed that the secondary structures of CA-MA in water solution were 17.4% alpha-helix and 82.6% random coil but no beta-sheet. Our results demonstrated that functional CA-MA can be produced in sufficient quantities using the ubiquitin fusion technique. This is the first report on the heterologous expression of a hybrid antibacterial peptide fused to ubiquitin in E. coli.     

Optimisation of expression and immobilized metal ion affinity chromatographic purification of recombinant (His)6-tagged cytochrome P450 hydroperoxide lyase in Escherichia coli

Fatty acid hydroperoxide lyase (HPL) is a cytochrome P450 acting on fatty acid's hydroperoxides in many plants. The optimisation of the expression of recombinant (His)(6)-tagged HPL in Escherichia coli is described: the highest HPL production yield were obtained with TB medium supplemented with 2.5 mM delta-aminolevulinic acid and 0.5 mM IPTG. For the first time, the time course expression of a plant P450 in a bench-scale fermentor is detailed and the amount of recombinant HPL production is 16.3 mg/l. The UV-Visible spectrum of the recombinant (His)(6)-tagged HPL have been recorded after a Ni(2+)-based affinity chromatography (IMAC).     

High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris

Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.     

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.     

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.     

Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His(6)-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.     

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).     

抗菌肽Cec4a的重组表达和抗菌活性研究*

目的:构建Cec4a的原核重组表达体系,通过诱导表达、酶切纯化获得重组蛋白,并检测产物的抗菌活性。方法:基于Cec4a的序列设计引物,克隆Cec4a基因的DNA片段。利用原核表达载体(pCold-SUMO)构建重组原核表达质粒,并将其转化到大肠杆菌C41(DE3)等感受态细胞,使用IPTG进行诱导表达。通过Ni-NTA亲和层析柱纯化,获得含有His-SUMO标签的重组Cec4a融合蛋白。在SUMO蛋白酶酶切后,再次使用Ni-NTA亲和层析纯化,得到目的蛋白,最后用鲍曼不动杆菌(ATCC19606)作为指示菌检测表达产物的抗菌活性。结果:成功构建pCold-SUMO-Cec4a原核表达质粒,测序分析其序列与预期结果一致。Cec4a融合蛋白表达量为42.8mg/L,纯化后的Cec4a重组蛋白对鲍曼不动杆菌的MIC为4 μg/mL。结论:通过原核表达,并经Ni-NTA亲和层析纯化,获得了具有抗菌活性的重组蛋白Cec4a,为研究Cec4a的生物活性、抗菌机制及应用奠定了基础。     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

Overexpression of the recombinant Enterobacter cloacae P99 AmpC beta-lactamase and its mutants based on a phi105 prophage system in Bacillus subtilis

AmpC beta-lactamase is a bacterial enzyme with great clinical impact as it mediates beta-lactam antibiotic resistance in many Gram-negative bacteria. To facilitate the structure-function relationship studies on this clinically important enzyme, we developed new strategies for production of recombinant Enterobacter cloacae P99 AmpC beta-lactamase in Bacillus subtilis. With the utilization of a special thermo-inducible phi105 phage system, functionally active AmpC beta-lactamase was expressed in B. subtilis, either in an extracellular native form or an intracellular N-terminal (His)(6)-tagged form. A higher expression level was achieved when expressing the enzyme as the intracellular (His)(6)-tagged protein rather than as the extracellular native protein. In addition, from the approach of producing intracellular tagged protein, highly pure (>95%) (His)(6)-tagged beta-lactamase wild-type and mutants (Y150C and K315C) were obtained after a one-step nickel affinity chromatography with a yield of 28.5, 66, and 0.85 mg/L of culture, respectively. Furthermore, the Y150C and K315C mutants were characterized so as to investigate the roles of the conserved residues, Tyr150 and Lys315, in the AmpC beta-lactamase. Severe impairment in hydrolytic abilities and restored secondary structures of the Y150C and K315C mutants suggested the major contribution of these two residues in the catalytic reaction rather than the structural framework in the AmpC enzyme.     

E. coli expression and purification of human and cynomolgus IL-15

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)₆-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His)₆-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3⁻ CD8⁺ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.     

Expression of secreted His-tagged S-adenosylmethionine synthetase in the methylotrophic yeast Pichia pastoris and its characterization, one-step purification, and immobilization

S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.     

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.     

High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.     

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.