The pAX plasmids: new gene-fusion vectors for sequencing, mutagenesis and expression of proteins in Escherichia coli

A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli. Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose. The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus. The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions. A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations. The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis. This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E. coli.     

提高Xa因子酶切效率的策略

为提高Xa因子对融合蛋白CBD-IGF和CBD-PACAP的酶切效率 ,以便高效释放非融合的重组多肽 ,利用基因工程技术在两个融合蛋白中Xa因子识别位点 (Ile-Glu-Gly-Arg↓ )前均引入 7个氨基酸组成的富含甘氨酸柔性短肽 (Gly-Thr-Gly-Gly-Gly-Ser-Gly)。纤维素亲和层析纯化各个融合蛋白 ,比较Xa因子对引入短肽前、后融合蛋白的酶切效率。比较结果表明 :短肽的引入不同程度地提高了融合蛋白CBD-IGF和CBD-PACAP对Xa因子的敏感性 ;但总体上CBD-IGF对Xa因子的敏感性比CBD-PACAP低。此研究结果提供了一种提高Xa因子酶切效率的策略。     

Cloning and expression in E. coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin

A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasmin was constructed and expressed in Escherichia coli as a fusion with beta-galactosidase. The gene was designed with a recognition site for the plasma protease, Factor Xa, coded for immediately prior to the N-terminus of caltrin. The beta-galactosidase-caltrin fusion protein was cleaved with Factor Xa to give caltrin, which was identified by its size on SDS-PAGE, its ability to react with an antiserum raised to the N-terminal nonapeptide of caltrin and its N-terminal amino acid sequence. After partial purification, synthetic caltrin was found to be active in an assay involving inhibition of growth of E.coli.     

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.     

Production in Escherichia coli of porcine type-I collagenase as a fusion protein with beta-galactosidase.

Porcine type-I collagenase (Colg-1) was produced as a fusion protein in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to Colg-1. Recombinant collagenase (reColg-1) was biologically active in the form of a fusion protein and could be released by treatment with factor Xa to yield Colg-1 with the authentic N terminus (phenylalanine) found in vivo. The results show that reColg-1 produced in E. coli is folded correctly, cleaves type-I collagen into 1/4 and 3/4 fragments at the characteristic Colg-sensitive site, and is produced at high enough levels to generate a source of recombinant enzyme for x-ray crystallography studies.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin

A gene which codes for the 66-residue polypeptide of kappa-bungarotoxin has been chemically synthesized by linking together 3 synthetic double-stranded oligonucleotides in a bacterial plasmid. The synthesis incorporated six unique silent restriction sites spaced throughout the gene for use in cassette mutagenesis. Direct expression of the kappa-bungarotoxin polypeptide by itself in Escherichia coli failed to result in a stable product. The toxin polypeptide was stabilized and expressed in E. coli as part of a fusion protein with rat intestinal fatty acid binding protein under control of the nalidixic acid inducible recA promoter. Two fusion protein constructs were prepared that differed only in the cleavage site between the fatty acid binding protein and the toxin polypeptide. One contained a factor Xa cleavage site, and the other, since the toxin itself is devoid of methionine, contained a methionyl residue that served as a cyanogen bromide cleavage site. The fusion proteins were isolated by ion-exchange chromatography and reverse-phase HPLC. The construct containing the factor Xa cleavage site could not be cleaved under nondenaturing conditions. On the other hand, kappa-bungarotoxin was efficiently cleaved from the methionyl fusion protein with CNBr. The toxin polypeptide was isolated by reverse-phase HPLC and ion-exchange chromatography and produced a complete and specific blockade of neuronal nicotinic acetylcholine receptors in chick ciliary ganglia which was indistinguishable from that produced by a comparable amount of venom-purified kappa-bungarotoxin.     

Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein

Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy. The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region. A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions. Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa. The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture. After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter. Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84).     

The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn(2+) flavoprotein

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.     

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.     

High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin

To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.     

Expression, purification, and kinetic characterization of recombinant rat cysteine dioxygenase, a non-heme metalloenzyme necessary for regulation of cellular cysteine levels

Cysteine dioxygenase (CDO, EC 1.13.11.20) is a non-heme mononuclear iron enzyme that oxidizes cysteine to cysteinesulfinate. CDO catalyzes the first step in the pathway of taurine synthesis from cysteine as well as the first step in the catabolism of cysteine to pyruvate and sulfate. Previous attempts to purify CDO have been associated with partial or total inactivation of CDO. In an effort to obtain highly purified and active CDO, recombinant rat CDO was heterologously expressed and purified, and its activity profile was characterized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility, and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The approximately 40.3 kDa full-length fusion protein was purified to homogeneity using a three-column scheme, the fusion tag was then removed by digestion with factor Xa, and a final column step was used to purify homogeneous approximately 23 kDa CDO. The purified CDO had high specific activity and kinetic parameters that were similar to those for non-purified rat liver homogenate, including a Vmax of approximately 1880 nmol min-1 mg-1 CDO (kcat=43 min-1) and a Km of 0.45 mM for L-cysteine. The expression and purification of CDO in a stable, highly active form has yielded significant insight into the kinetic properties of this unique thiol dioxygenase.     

Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin.

Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.     

A bifunctional chimeric protein consisting of MutS and beta-galactosidase

A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for beta-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 microl volumes after 120 and 15 min incubation, respectively.     

An import-competent precursor of small subunit of ribulose-1,5-bisphosphate carboxylase generated by factor Xa cleavage from a beta-galactosidase fusion expressed in Escherichia coli.

A plasmid-encoding fusion protein interlinked by factor Xa recognition sequence between beta-galactosidase and a precursor of the small subunit of wheat ribulose-1,5-bisphosphate carboxylase has been constructed. The plasmid directed abundant synthesis of the fusion protein in Escherichia coli. The recombinant protein was accumulated in an aggregated form that was associated with the bacterial membranes. A procedure was developed to isolate the fusion protein in a relatively pure and soluble form. Bovine factor Xa cleaved the isolated chimera to generate the complete chloroplast precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase from the fused beta-galactosidase. The cleaved precursor protein was imported into the isolated chloroplasts and processed to yield its mature counterpart.     

Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233)

To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies.     

The N-terminal 21 amino acids of a 70 kDa protein of the yeast mitochondrial outer membrane direct E. coli beta-galactosidase into the mitochondrial matrix space in yeast cells

The intracellular location of fusion proteins was investigated in yeast cells. They consisted of the N-terminal 21, 61 or 292 amino acids of the 70 kDa protein of the yeast mitochondrial outer membrane and an enzymatically active E. coli beta-galactosidase. The hybrids containing 61 or 292 residues of the 70 kDa protein, as well as the original 70 kDa protein, were localized on the outer membrane in a tightly membrane-bound form. In contrast, the other hybrid was exclusively localized in the mitochondrial matrix space as a soluble protein.     

A mechanism of protein localization: the signal hypothesis and bacteria

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.     

The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase.

The macrophage-induced gene (mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown. Here we have characterized the Mig protein by biochemical methods. A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli. Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product. The sequence of the fusion gene and of the parent gene in M. avium were reexamined. This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before. The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases. Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E. coli. These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay. Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates. Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases. We postulate a novel medium-chain acyl-CoA synthetase motif. We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.     

Purification of recA-based fusion proteins by immunoadsorbent chromatography. Characterization of a major antigenic determinant of Escherichia coli recA protein

Monoclonal antibodies to Escherichia coli recA protein were prepared, characterized, and used as affinity reagents for the purification of recA and recA:somatostatin fusion proteins. The monoclonal antibodies recognize an antigenic determinant or determinants located between amino acids 260 and 330 of recA. Addition of a fragment of the recA gene coding for these amino acids to an unrelated gene (beta-galactosidase) allowed the resulting beta-galactosidase fusion protein to be recognized by the recA monoclonal antibodies.