Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: preparation for a functional analysis

Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0M urea in 0.5M increments. Purified RHG1 protein obtained from the 1.5 and 7.0M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0M urea sample, whereas refolding from the 1.5M urea sample yielded 0.2mg/ml protein. The 7.0M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.     

High level expression and purification of active recombinant human interleukin-8 in Pichia pastoris

Human interleukin-8 (hIL-8) is a member of interleukin family which functions as a chemotactic factor as well as an angiogenesis mediator. Previously, a study reported that hIL-8 could be purified from inclusion bodies using a prokaryotic expression system, however, the required re-naturation step limits the recovery of fully active protein. In this study, soluble recombinant hIL-8 was expressed as a secreted protein at high level in Pichia pastoris under the control of AOX1 (alcohol oxidase 1) promoter. A simple purification strategy was established to recover rhIL-8 from the fermentation supernatant. The process includes precipitation with 80% saturation ammonium sulfate and CM Sepharose ion exchange chromatography, yielding 30 mg/L purified rhIL-8 at over 95% purity. The obtained rhIL-8 displays high specific activity, stimulating the migration of mouse neutrophils at concentrations as low as 0.25 ng/mL. Our results demonstrate that P. pastoris expression system is an efficient tool for large-scale manufacture of active recombinant hIL-8 for various applications.     

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.     

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K_D ∼ 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Escherichia coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP.     

A simple novel method for the preparation of noncovalent homodimeric, biologically active human interleukin 10 in Escherichia coli-enhancing protein expression by degenerate PCR of 5' DNA in the open reading frame

DNA inserts encoding human interleukin 10 (hIL-10), optimized for codon usage and secondary RNA structure, were purchased from several commercial sources and subcloned into a pMon vector. Despite the optimization, protein expression was nil. We therefore subjected the 5′ segment of the cDNA encoding N-terminal amino acids 2–11 to degenerate PCR in order to create a small library of 130 K theoretical cDNA combinations that would not change the respective amino acid sequence and tested their expression. After screening over 320 colonies 10 hIL-10 clones encoding the original amino acid sequence were identified. Three nucleotide substitutions were sufficient to ensure reasonable protein expression. Subsequently, hIL-10 was expressed in Escherichia coli, refolded and purified to homogeneity, yielding over 95% electrophoretically pure noncovalent homodimeric protein, which was biologically active in MC/9 cells. The yield of recombinant hIL-10 from 10 L of fermentation culture was 60 mg and a protocol for its long-term storage as a carrier-free lyophilized powder at −20° was developed.     

Cytoplasmic and periplasmic expression of a highly basic protein,human interleukin 4, inEscherichia coli

Summary Human IL-4 (hIL-4) has been cloned from a human T cell line based on its homology to the murine IL-4 cDNA sequence [36]. We have compared cytoplasmic and extra-cytoplasmic expression of this basic protein inEscherichia coli using various combinations of promoters, replicons and host strains. Strains producing a cytoplasmic product were most successful at heterologous protein expression, producing up to 500 mg/l of an inactive aggregated form of the protein. The biological activity of the protein could be restored by refolding the protein with guanidine hydrochloride and glutathione giving a specific activity identical to that of IL-4 derived from CHO cell lines stably transformed with an hIL-4 expression plasmid. Strains designed to secrete human IL-4 into the periplasmic space produced far less protein (approximately 5 mg/l). However, a significant fraction of this protein was detected in the culture medium. This fraction appeared to be soluble after ultracentrifugation, and demonstrated high specific activity without refolding. Leakage of heterologous protein into the culture medium may be a viable way to recover biologically active products without relying on the denaturation and refolding in vitro that can, at times, yield incorrectly folded gene product.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.     

Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system

In this work, the intein fusion approach was used for expression and purification of cathelicidin-like peptide SMAP-29 from Escherichia coli cultures. To overcome the high toxicity of the antimicrobial peptide against host cells, both C- and N-terminal fusions with Sce VMA intein were evaluated. The fusion of SMAP-29 with the N-terminus of intein had a dramatic lethal effect. In contrast, chimeric constructs harboring SMAP-29 linked to the C-terminus of intein displayed no significant inhibition of bacterial growth. Expression of intein-SMAP fusion protein was then induced in ER2566 E. coli strain by IPTG addition and different experimental conditions were tested in order to optimize the recovery of the soluble protein complex. Peptide purification was carried out by affinity chromatography: the chitin binding domain linked to intein was used to immobilize the chimeric protein on a chitin column and intein-mediated splicing of target peptide was obtained by thiol addition. Microbroth dilution assay showed that recombinant SMAP-29 displayed a high, dose-dependent bactericidal activity. These data demonstrate that the fusion of SMAP-29 with C-intein was able to inactivate the antimicrobial properties of the cathelicidin peptide allowing the expression of fusion protein in the host cell. The intein-mediated purification supplied an effective way to recover the fusion partner in its proper biologically active form.     

Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide

The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni²⁺-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni²⁺-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.     

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.     

A two-plasmid Escherichia coli system for expression of Dr adhesins

This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.     

Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli

Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli.     

Expression and Purification of Biologically Active Rat Bone Morphogenetic Protein-4 produced as Inclusion Bodies in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Rat bone morphogenetic protein-4 (rBMP-4) cDNA was cloned from rat osteoblasts by RT-PCR and expressed in E. coli. Monomeric, dimeric and polymeric forms of recombinant rat BMP-4 (rrBMP-4) were obtained from inclusion bodies after solubilization with urea. The dimer was separated from the remaining polymer and host cell contaminants using size exclusion chromatography. Furthermore, purified rrBMP-4 was stabilized at low urea concentration (40 mm) and at pH 8.5 through the addition of bovine serum albumin. Both, rrBMP-4 dimer and polymer were biologically active as tested by the induction of alkaline phosphatase activity in MC3T3-E1 cells.     

Expression of a human proenkephalin a cDNA inEscherichia coli

A 1000 base pair cDNA coding for the entire human proenkephalin A(proA) polypeptide was subcloned into the multifunctional pMPV 2911/ME. coli vector. The recombinant plasmid was found to express an approximately 30 kDa prohormone, which was recognized by a Met-Arg⁶-Phe² antibody, directed against the C-terminal part of the enkephalin A prohormone. The expression of human proenkephalin A cDNA should thus permit the rapid purification of unfused recombinant enkephalin A prohormone, which itself may provide a model substrat to identify endoproteolytic processing activities.     

HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His₆Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.     

Purification and Properties of a Ribonuclease from Cowpea Cotyledons

The isolation and characterisation of cotyledonary ribonucleases (RNase; EC 3.1.27.1), are basic steps to understand the physiology and biochemistry of RNA turnover and mobilisation during seed germination and seedling establishment, as well as how environmental stresses affect them. RNase was isolated and purified 928-fold, to apparent electrophoretic homogeneity from 5-d-old seedlings of Vigna unguiculata. It is a protein with an apparent molecular mass of 16 kDa having three major isoforms. Its optimum pH is 5.8, which decreases to 5.2 in presence of KCl. It has an apparent K_m of 0.80 mg RNA cm^-3 and retains 40 % of its activity when heated to 80 °C. It is completely inhibited by Cu²⁺, Hg²⁺ and Zn²⁺ and is almost insensitive to Mg²⁺, Ca^2+- and EDTA. Urea, Fe²⁺, Co²⁺ and 2-mercaptoethanol partially inhibit its activity. Its amino acid composition shows a resem lance to that of other plant RNases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.     

Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization

The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and overexpressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 °C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 °C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 °C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.     

Purification and partial characterization of two extracellular keratinases of Scopulariopsis brevicaulis

Two extracellular keratinases of Scopulariopsis brevicaulis were purified and partially characterized. The enzymes were isolated by the techniques of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These keratinases (K I & K II) were purified approximately 33 and 29 fold, respectively. SDS-PAGE of the products of gel filtration chromatography (K I & II) produced only one band each, suggesting homogeneity. The optimum pH for both keratinases was 7.8, while the optimum temperatures were 40°C (K I) and 35°C (K II). Estimated molecular weights were 40–45 KDa and 24–29 KDa for K I & K II respectively. Both keratinases were inhibited by phenylmethylsulfonyl fluoride which suggests a serine residue at or near an active site.     

Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies

Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs), making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain (CD), a complete understanding of its catalytic regulation is unavailable. Also unavailable are atomic or near-atomic resolution information about their interactions, often weak or transient, with regulating phosphatases and downstream targets. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. Through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, we have achieved the ability to bacterially produce large quantities of the isotopically-labeled CD of c-Src, the prototypical SFK, and of its activating Tyr-phosphorylated form. All constructs produce excellent spectra allowing solution NMR studies of this family in an efficient manner. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.