Preparation of recombinant glycoprotease of Pasteurella haemolytica A1 utilizing the Escherichia coliα-hemolysin secretion system

Abstract Three murine monoclonal antibodies were prepared against the recombinant glycoprotease of Pasteurella haemolytica A1 expressed in Escherichia coli . These monoclonal antibodies were able to recognize the authentic glycoprotease from P. haemolytica A1 culture supernatant. A recombinant plasmid which contained most of the glycoprotease gene of P. haemolytica A1 fused with the secretion signal sequence from hlyA of the E. coli α-hemolysin determinant was constructed. This recombinant plasmid expressed a fusion protein (Gcp-F) which was secreted into the culture supernantant by E. coli cells when the α-hemolysin secretion functions HlyB and HlyD are supplied in trans. Gcp-F could be readily recovered from the supernatant free from other cellular materials and is suitable for use in vaccine trials and challenge experiments in animals.     

Refolding and characterization of recombinant human GST-PD-1 fusion protein expressed in Escherichia coli

Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.     

Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene.

Pasteurella haemolytica serotype A1 secretes a glycoprotease which is specific for O-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pH1, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPH1. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPH1. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orfX, whose product is believed to function in the regulation of macromolecule biosynthesis.     

Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli

OsNifU1A is a NifU-like rice (Oryza sativa) protein, discovered recently. Its amino acid sequence is very homologous to the sequence of cyanobacterial CnfU and to the sequences of NifU C-terminal domains. Based on its sequence, OsNifU1A is probably a modular structure consisting of two CnfU-like domains, with domain I (formed by residues Leu73 to Gly153) and domain II (formed by residues Leu154 to Ser226). Domain I have a conserved Cys-X-X-Cys motif, which may function as an iron-sulfur cluster assembly scaffold. Domain II lacks a Cys-X-X-Cys motif and therefore, cannot function analogously. Other NifU-like proteins, with sequences homologous to OsNifU1A domain II, have been identified during plant genomic projects; however, the biological roles of these domains remain unknown. We successfully constructed an Escherichia coli expression system for OsNifU1A domain II that enabled us to synthesize and purify milligram quantities of protein for use in structural and functional studies. Using the Gateway system, we built DNA sequences corresponding to two OsNifU1A domain II fusion proteins. One construct has a (His)6 sequence upstream of the OsNifU1A domain II sequence; the other has an upstream thioredoxin-(His)6 sequence. Recombinant OsNifU1A domain II fusion proteins were extracted from E. coli inclusion bodies by dissolving them in 6 M guanidine-HCl. About 36% of the total (His)6/OsNifU1A domain II fusion protein initially present remained soluble after guanidine-HCl was completely removed by step-wise dialysis; whereas, recovery of soluble Trx-(His)6 fusion protein was about 60% of the total cell lysate. About 2 mg of 15N-labeled OsNifU1A domain II was purified for NMR spectral studies. Examination of the OsNifU1A domain II 1H-15N HSQC NMR spectrum indicated that the purified protein was monomeric and correctly folded. Therefore, we established an efficient procedure for synthesis and purification of 15N-labeled OsNifU1A domain II in quantities sufficient for heteronuclear NMR solution structure studies.     

Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies

We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli. A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag. The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5. The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity. In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein. Both the Vmax (0.46 micromol. min(-1). mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1. Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa). As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers. The purified enzyme was soluble at concentrations up to 100 microg/ml. A further increase of protein concentration resulted, however, in precipitation of the enzyme.     

Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli.

CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay.     

Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli

A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates.     

A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity

omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons. Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia. In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function. The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA. The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC). The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40 mg/L. The analgesic function was detected by using mouse hot-plate assay. After intracranially administering fusion protein with the dose of 0.6 mg/kg, marked analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6 h.     

Purification of recombinant ribonuclease T1 expressed in Escherichia coli

A protocol for the rapid purification of ribonuclease T1 expressed from a chemically synthesized gene cloned into Escherichia coli is described. QAE ion-exchange and Sephadex G-50 chromatography are used to give over 300 mg (88% yield) of pure ribonuclease T1 from 61 of liquid culture in 3 days. We also report a new absorption coefficient for RNase T1: E1%278 nm = 15.4.     

The glycoprotease of Pasteurella haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin.

HL-60 cells and neutrophils treated with the glycoprotease from Pasteurella haemolytica A1, an enzyme which is specific for O-sialoglycoproteins, were found to be incapable of binding P-selectin but still bound E-selectin. Comparative analysis of [35-S] cysteine labeled proteins from HL-60 cells by 2-dimensional electrophoresis indicated that two major proteins with M(r) 100 and 115 kd were significantly removed from cells which had been treated.     

A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli

The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale. Many peptides need carboxy amidation for full activity or prolonged bioavailability. However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity. Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system. This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents. Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide. With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.     

Refolding and purification of human papillomavirus type 16 E7-lacZ fusion protein expressed in Escherichia coli.

Human papillomavirus (HPV) type 16 E7-lacZ fusion protein was produced in Escherichia coli, extracted as inclusion bodies, refolded with reducing reagents, and subjected to gel filtration. The refolded protein was purified by ion-exchange column chromatography, resulting in a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1H nuclear magnetic resonance spectral changes were observed in the high field methyl region in the presence of Zn2+ ion, suggesting that the refolded form of the fusion protein is possibly renaturated into the putative zinc finger motif (C. Edmond and K. H. Vousden, 1989, J. Virol. 63, 2650-2656) and supporting the data of J. A. Rawls, R. Pusztai, and M. Green (1990, J. Virol. 64, 6121-6129) on zinc binding to E7 protein using radioisotopically labeled zinc ion.     

A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli.

The high affinity binding interaction of biotin to avidin or streptavidin has been used widely in biochemistry and molecular biology, often in sensitive protein detection or protein capture applications. However, in vitro chemical techniques for protein biotinylation are not always successful, with some common problems being a lack of reaction specificity, inactivation of amino acid residues critical for protein function and low levels of biotin incorporation. This report describes an improved expression system for the highly specific and quantitative in vivo biotinylation of fusion proteins. A short 'biotinylation peptide', described previously by Schatz, is linked to the N-terminus of Escherichia coli thioredoxin (TrxA) to form a new protein, called BIOTRX. The 'biotinylation peptide' serves as an in vivo substrate mimic for E. coli biotin holoenzyme synthetase (BirA), an enzyme which usually performs highly selective biotinylation of E.coli biotin carboxyl carrier protein (BCCP). A plasmid expression vector carrying the BIOTRX and birA genes arranged as a bacterial operon can be used to obtain high level production of soluble BIOTRX and BirA proteins and, under appropriate culture conditions, BIOTRX protein produced by this system is completely biotinylated. Fusions of BIOTRX to other proteins or peptides, whether these polypeptides are linked to the C-terminus or inserted into the BIOTRX active site loop, are also quantitatively biotinylated. Both types of BIOTRX fusion can be captured efficiently on avidin/streptavidin media for purification purposes or to facilitate interaction assays. We illustrate the utility of the system by measurements of antibody and soluble receptor protein binding to BIOTRX fusions immobilized on streptavidin-conjugated BIAcore chips.     

pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli

Glutathione-S-transferase (GST) fusion protein expression vectors are often employed for the expression and purification of proteins in Escherichia coli. GST is then removed by site-specific proteolysis using thrombin. However, the presence of internal thrombin cleavage sites in expressed proteins can severely affect the purification of intact proteins. Cysteine-dependent aspartate-specific proteases (caspases) are efficient enzymes with defined substrate specificity. Unlike most of the proteases used for the removal of affinity tags, caspases do not leave any amino acids at the amino-terminus of cleaved proteins. We have engineered the caspase-6 site VEMD in a pGEX vector to give the pC6-2 vector. The caspase-6 can be easily removed after cleavage. Here, we describe the detailed protocol for purifying proteins using our pC6-2/caspase-6 expression and purification system. The cleavage by caspase-6 occurs in <30 min and the entire procedure can be completed in 2 d.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Purification of an L-asparaginase-atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.     

Refolding and activation of human prorenin expressed in Escherichia coli: application of recombinant human renin for inhibitor screening

Human prorenin was expressed in Escherichia coli as a fusion protein of thioredoxin. The chimeric protein, which accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by an arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the recombinant human renin activity. Using the recombinant human renin, we screened its inhibitory activity in fermented soybean paste (miso) and demonstrated that miso contained renin inhibitory activity derived from soybean. The IC(50) values for soybean and steamed soybean extracts were determined to be 1.9 and 1.6 mg/ml, respectively. This is the first demonstration of renin inhibitory activity in miso and soybean.     

Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock

A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and 25% sucrose; the low osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and the heat-treatment temperature of 100 degrees C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.     

Growth-phase regulation of the Escherichia coli thioredoxin gene

The two promoters of Escherichia coli trxA gene were separately cloned into pKO100 as well as pJEL170. Galactokinase expression in cells containing the pKO100 derivatives was found to be negatively correlated with growth rate and was 6- to 20-fold higher in stationary cultures than in exponential cultures. The expression of trxA-galK was induced by amino acid starvation in a RelA(+) strain but not in an isogenic Rel(-) strain indicating that the control involves guanosine 3',5'-bispyrophosphate (ppGpp). RpoS, which appears to be essential for expression of most stationary phase expressed genes, is not required for trxA expression. Increased expression of relA, which increases ppGpp concentration, increases trxA expression.