Overexpression and purification of recombinant atrial natriuretic peptide using hybrid fusion protein REF-ANP in Escherichia coli

Atrial natriuretic peptide (ANP), a small peptide consisting of 28 amino acids, has been applied in clinical treatment for heart failure, but it can encounter proteolytic degradation during its expression in host cells. Therefore, it is usually reported that ANP was expressed as a part of fusion protein. The aim of our study was to use an overexpression system to express the fusion protein REF-ANP and to optimize a purification method. First, Escherichia coli DH5alpha was transformed with constructed expression vector containing two tandem copies of ref-anp gene and the fusion protein REF-ANP was overexpressed in shaking flask culture. Subsequently, the inclusion bodies were purified with reverse phase chromatography and pooled fractions were lyophilized. After this step, REF-ANP can be solubilized under native conditions without urea. After cleavage reaction, the sample was subjected to size exclusion chromatography and then rANP was polished with reverse phase chromatography. The final purity of rANP was more than 98% and the recovery of rANP per liter of shaking flask culture was more than 3mg. Such methods as mass spectrometry, capillary isoelectrofocusing analysis, and N-terminal amino acid sequence were used to identify rANP. The capillary isoelectrofocusing analysis showed that the pI of ANP was about pH 9.7. In this study, an efficient refolding and purification process should make scaling-up procedures easier and more successful than earlier reports. Moreover, it is possible that the refolding and purification method along with the overexpression system described in this article may offer new ideas on optimizing expression and purification of other kinds of short peptides.     

Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli

Overexpression of rhIFN-alpha2b was obtained by synthesizing a codon optimized gene for IFN-alpha2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNalpha, which had the IFN-alpha2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible lambdaP(L) promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2g/L at a final OD(600) of 67. At this point, the IBs represented approximately 40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of approximately 3g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of approximately 3 x 10(9)IU/mg protein.     

Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.     

Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form

Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase. The E. coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus. The plasmid was designated pAasH. The activity of Aas in membranes was assessed from several cell lines. Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl beta-d-thiogalactopyranoside (IPTG) or lactose, respectively. When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid. Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography. The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body.     

Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli

Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.     

大肠杆菌holo-ACP的过表达、分离纯化及长链脂酰ACP的合成

[目的]获得高纯度大肠杆菌holo-ACP和多种长链脂酰ACP,为研究细菌脂肪酸、类脂A和N-酯酰高丝氨酸内脂等物质的合成提供底物.[方法和结果]采用PCR方法扩增得到大肠杆菌酰基载体蛋白基因(acpP)和holo-ACP合成酶基因(acpS).使用载体pBAD24、pBAD34和pET28b分别克隆了acpP和acpS,得到pBAD-ACP、pET-ACP和pET-ACP-ACPS 3个ACP表达质粒和一个AcpS表达质粒pBAD-ACPS.分别用3个ACP表达质粒转化大肠杆菌DH5a和BL21(DE3),构建了DH5αpBAD-ACP、BL21(DE3)/pET-ACP和BL21(DE3)/pET-ACP-ACPS 3种ACP生产菌株.与holo-ACP纯化常用菌株DK574相比,虽然三菌株在诱导时均能过量表达ACP,但是holo-ACP所占比例偏低.为了提高ACP生产菌株holo-ACP的产量,用质粒pBAD-ACPS分别转化上述3种ACP生产菌株,获得了3种携带双质粒的ACP生产菌株.表达结果显示携带pBAD-ACP和pBAD-ACPS双质粒的DH5a菌株比DK574菌株能产生更多的holo-ACP,且纯度也得到提高(纯度达99%).同时使用UNOsphere Q阴离子交换层析从这一菌株培养物中分离纯化到了高纯度的holo-ACP,并以纯化到的holo-ACP和多种长链脂肪酸为底物在哈氏弧菌脂酰ACP合成酶的催化下,合成了多种长链脂酰ACP.[结论]通过研究获得一株holo-ACP高产菌株,并证明在大肠杆菌菌株中,同时表达acpP基因和acpS基因,有利于holo-ACP的产生.     

Overexpression and rapid purification of Escherichia coli formamidopyrimidine-DNA glycosylase

Formamidopyrimidine DNA glycosylase (Fpg) is a DNA glycosylase with an associated AP lyase activity. As a DNA repair enzyme, Fpg excises several modified bases from DNA associated with exposure to oxidizing agents such as free radicals. Experiments in many laboratories have been limited by the availability of the enzyme, and its production required at least a week of work to complete its purification. We have devised a new method that decreases the time and expense of purification of Fpg that should render this protein accessible to any laboratory. Fpg was subcloned into a gamma P(L) promoter-containing vector (pRE) and overproduced in the appropriate Escherichia coli host cells to about 25% of the total cellular protein. Fpg was purified to homogeneity in a simple two-step procedure with a 50% saving in time when compared to the previously known procedure. Comparative studies showed that the excision of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine, and to a lesser extent, 8-hydroxyadenine was virtually identical for the Fpg purified using this method and for the Fpg purified by the original method. Therefore, this method should prove useful for a large number of laboratories and further research on oxidative DNA damage.     

Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm

Present research provided an efficient approach to obtain large quantities of active recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm, Bombyx mori. The cDNA encoding mature CI-b1 was cloned into pDEST17 vector. Recombinant protein with hexa-histidine tag attached to the N-terminal of CI-b1 was expressed in Escherichia coli Origami B cells. It can be purified to homogeneity via the gel filtration chromatography on a Sephacryl S-200 column followed the affinity chromatography on a Ni-NTA column. The two sequential purification procedures yielded 4.3mg purified (His)(6)-tagged CI-b1 from 200ml of culture medium. Studies on (His)(6)-tagged CI-b1 revealed that three disulfide bonds were formed in the recombinant CI-b1 and the inhibitory properties of recombinant CI-b1 against alpha-chymotrypsin were similar to those of native CI-b1. Recombinant CI-b1 immobilized on Ni-NTA resin was used to detect the interactions occurring between the CI-b1 and its target factors.     

Expression and purification of recombinant hemoglobin in Escherichia coli

Background

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.

Methodology/Principal Findings

As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.

Conclusion/Significance

Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.     

Expression and purification of recombinant neurotensin in Escherichia coli

An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques.     

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19–405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni–NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 μg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 μg/ml.     

Overexpression and purification of asparagine synthetase from Escherichia coli.

Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.     

Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli.

Rat liver protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds. We have developed an efficient method for its overproduction in Escherichia coli. Using a T7 RNA polymerase expression system, isolated yields of 15-30 mg/liter of recombinant rat PDI are readily obtained. Convenient purification of the enzyme from E. coli lysates involves ion-exchange (DEAE) chromatography combined with zinc chelate chromatography. The recombinant PDI shows catalytic activity identical to that of PDI isolated from bovine liver in both the reduction of insulin and the oxidative folding of ribonuclease A. The enzyme is expressed in E. coli as a soluble, cytoplasmic protein. After complete reduction and denaturation in 6 M guanidinium hydrochloride, PDI regains complete activity within 3 min after removal of the denaturant, implying that disulfide bonds are not essential for the maintenance of PDI tertiary structure. Both the protein isolated from E. coli and the protein isolated from liver contained free cysteine residues (1.8 +/- 0.2 and 1.4 +/- 0.3 SH/monomer, respectively).     

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.     

Expression and purification of recombinant human alpha-defensins in Escherichia coli

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.     

Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis and capable of eliciting protective antibodies in infected hosts, and therefore has potential as a candidate vaccine to prevent infection with this significant human pathogen. The recombinant MOMP clone, L2rMOMP, contained the entire MOMP gene including the encoded leader sequence. Large quantities of chlamydial MOMP were expressed, some of which was processed and translocated to the E. coli surface. Surface localization of the MOMP was demonstrated by the binding of anti-MOMP monoclonal antibodies to the surface of the induced clone, and was visualized by fluorescence and electron microscopy. The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune response.     

Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co²⁺-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl₂ and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm² each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.     

Expression, purification and characterization of the Escherichia coli integral membrane protein YajC

Escherichia coli YajC is a small integral membrane protein with a single transmembrane helix. The gene yajC is part of the secD operon and the protein is identified in the SecDF-YajC complex. However, the exact function of YajC remains a mystery. While its function is usually discussed in the context of the SecDF-YajC complex, studies have shown that SecD/F, rather than YajC, are essential for those functions. Recently YajC is identified as the mysterious protein that co-crystallized with AcrB. To further investigate the structure of YajC, we expressed and purified the protein in a detergent solubilized state. The protein assumed a folded structure containing mixed α/β secondary structures, consistent with the structural prediction. Using signal Cys mutations and thiol-specific probes, we found the C-terminus of YajC was cytoplasmic, while the N-terminus of YajC was buried in the membrane. In addition, we expressed and purified a truncated fragment of YajC that corresponded to the C-terminal cytoplasmic domain (YajC(CT)). YajC(CT) formed a compact structure rich in β-strands and existed as a trimer.     

Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.     

Production and purification of recombinant human oxytocin overexpressed as a hybrid protein in Escherichia coli

The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.