A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies

A recombinant fusion protein consisting of native Escherichia coli heat-stable enterotoxin (STa) and a dimer of a synthetic IgG-binding fragment (ZZ), derived from Staphylococcus aureus protein A was produced in E. coli. The fusion protein (ZZSTa) was secreted in large quantities into the growth medium and recovered by affinity chromatography on IgG-Sepharose. Rabbits immunized with the fusion protein responded by producing high serum levels of anti-STa antibodies that also effectively neutralized STa toxicity in infant mice. The fusion peptide ZZSTa had a substantially decreased toxicity as compared with native STa. A polymeric form of ZZSTa separated by size fractionation was about 100 times less toxic than the monomeric fusion protein, yet both forms had the same capacity to induce neutralizing antibodies. This suggests that modified non-toxic forms of ZZSTa with retained immunogenicity may be produced and tested for their usefulness as functional components in a vaccine against diarrhoea caused by enterotoxigenic E. coli.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

日本血吸虫中国大陆株22.6kD膜相关蛋白基因的克隆及其在大肠杆菌中的高效表达

以日本血吸虫ｍＲＮＡ为模板,用ＲＴＰＣＲ法快速克隆到一大小约６００ｂｐ的ＤＮＡ片段,ＤＮＡ序列分析证实,所扩增到的ＤＮＡ片段中含有日本血吸虫２２６膜相关蛋白（Ｓｊ２２６（Ｃｈ））基因,将该基因重组到表达型质粒ｐＧＥＸ４Ｔ中,表达的ＧＳＴ融合蛋白分子量约４８ｋＤ,用谷胱甘肽琼脂糖凝胶亲和层析柱纯化的重组蛋白不仅纯度好,而且得率高,纯化产量可达４０ｍｇ／Ｌ培养物,免疫试验结果表明该重组蛋白具有良好的抗原性,为其在血吸虫病抗感染中的免疫作用研究创造了条件。     

A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody

A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK). The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA. An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl. SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.     

日本血吸虫中国大陆株TPI基因的克隆及其表达产物特性

磷酸丙糖异构酶（ＴＰＩ）是血吸虫病疫苗重要的候选抗原基因之一。参考日本血吸虫已发表的ＴＰＩｃＤＮＡ序列,以日本血吸虫中国大陆株成虫ｍＲＮＡ为模板,用ＲＴ－ＰＣＲ法快速克隆出一大小约８００ｂｐ的ＤＮＡ片段。ＤＮＡ序列分析证实,所扩增到的ＤＮＡ片段即为日本血吸虫中国大陆株磷酸丙糖异构酶（ＳｊＴＰＩｃ）基因。将该基因重组到表达型质粒ｐＧＥＸ－４Ｔ中,表达的ＧＳＴ融合蛋白分子量约５４ｋＤ。用谷胱甘肽琼脂糖凝胶亲和层析柱纯化的重组蛋白不仅纯度好,而且得率高,纯化产量可达３０ｍｇ／Ｌ培养物。免疫试验结果表明该重组蛋白具有良好的抗原性,是一种较为理想的抗原分子。     

Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli.

The most frequently occurring kringle 4 domain of human apolipoprotein (a), Kringle 4-subtype 2 (K4(2)), was expressed as a fusion protein with the maltose binding protein in Escherichia coli using the "tac" promoter. Although the fusion protein was expressed without a signal sequence, 25% was secreted into the periplasmic space; the remainder was found associated with the soluble cytosolic fraction. The fusion protein was readily isolated from whole cell lysate by amylose agarose affinity chromatography. Although a factor Xa cleavage site was engineered into the fusion protein, it was found that release of the K4(2) protein was most conveniently achieved by proteolysis with subtilisin A. The cleavage product produced in this way was shown to be intact K4(2) with only the first three amino acid residues of the leading flanking peptide missing, as judged by N-terminal sequence analysis. K4(2) was isolated from the hydrolysate by FPLC on a Mono-Q column with a yield of 170 +/- 30 micrograms/g wet cells. The resulting protein was monomeric in phosphate-buffered saline as judged by size-exclusion chromatography and appeared to be folded as shown by spectroscopic and immunological assays. The recombinant K4(2) did not bind to either lysine- or proline-Sepharose, suggesting that the ligand binding activities of lipoprotein (a) may reside in the other kringle domains of apolipoprotein (a).     

Secretion of heterologous gene products to the culture medium of Escherichia coli.

Different constructs containing fragments of the Staphylococcal protein A gene have been introduced in Escherichia coli and the effect on expression and translocation of the various heterologous gene products have been studied. By reversing the orientation of the different protein A gene constructions in the plasmid vector, a dramatic 20-fold difference in expression was obtained, accompanied with secretion of the gene product to the culture medium. Similar results were obtained by "heat-shock" treatment of the E.coli host cells. These results suggest the presence in the protein A gene of a stress induced promoter, functional in E.coli. The system was used to efficiently secrete a fusion protein consisting of a protein A fragment and human insulin-like growth factor I (IGF-I) to the culture medium of E.coli HB101. The fusion protein was purified from the culture medium by IgG affinity chromatography in a one-step procedure giving more than 95% yield.     

Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin. Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation.

We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8258-8262). While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult. We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH. We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor.     

The purification of shikimate dehydrogenase from Escherichia coli.

A procedure was developed for the purification of shikimate dehydrogenase from Escherichia coli. Homogeneous enzyme with specific activity 1100 units/mg of protein was obtained in 21% overall yield. The subunit Mr estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 32 000. The native Mr, estimated by gel-permeation chromatography on a TSK G2000SW column, was also 32 000. E. coli shikimate dehydrogenase is therefore a monomeric NADP-linked dehydrogenase.     

B型肉毒毒素受体结合区C片段在大肠杆菌中的表达及免疫原性研究

目的:在大肠杆菌中表达、纯化B型肉毒毒素受体结合区C片段(BHc-C),研究其免疫原性。方法:将BHc-C基因克隆到原核表达载体pGEX-4T-1中,转化大肠杆菌BL21(DE3),经IPTG诱导表达GST-BHc-C融合蛋白并通过亲和纯化;以纯化的融合蛋白免疫BALB/c小鼠制备免疫血清,采用ELISA检测免疫血清的效价并测定其抗B型肉毒毒素中和活性。结果:在大肠杆菌中表达了GST-BHc-C融合蛋白;以该融合蛋白免疫小鼠获得高效价免疫血清,且该免疫血清具有中和活性。结论:获得了GST-BHc-C融合蛋白,并证实其具有免疫原性。     

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains.

A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form. The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc. Natl Acad. Sci. USA, 86, 3060-3064) who observed a substantial decrease in the yield of active enzyme when folded in the presence of a large C-terminal fragment. The fusion protein [DHFR(Cys152Glu)--Ile--DHFR (Met1Gln)] was efficiently expressed in E. coli cells and has an activity which is twice that of the wild-type enzyme in the standard assay. The Michaelis constants of the fusion protein for the substrate, dihydrofolate and the cofactor, NADPH, are essentially unchanged from those of the wild-type protein. The urea-induced in vitro unfolding reaction of the fusion protein at low concentrations was found to be fully reversible and follow a three state model, suggesting that the two domains unfold independently. At higher protein concentrations the unfolding transition broadened and shifted to a higher urea concentration. Size-exclusion chromatography results are consistent with the formation of aggregates at the higher protein concentration, even in the absence of denaturant.     

Recombinant Production, Isotope Labeling and Purification of ENOD40B: A Plant Peptide Hormone

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.     

Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes.

Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthesis of the K5 polysaccharide. A DNA fragment containing kfiD was amplified by PCR and cloned into the gene fusion vector pGEX-2T to generate a GST-KfiD fusion protein. The fusion protein was isolated from the cytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced recombinant bacteria by affinity chromatography and cleaved with thrombin. The N-terminal amino acid sequence of the cleavage product KfiD' corresponded to the predicted amino acid sequence of KfiD with an N-terminal glycyl-seryl extension from the cleavage site of the fusion protein. Anti-KfiD antibodies obtained with KfiD' were used to isolate the intact KfiD protein from the cytoplasms of E. coli organisms overexpressing the kfiD gene. The fusion protein, its cleavage product (KfiD'), and overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein could thus be characterized as a UDPglucose dehydrogenase.     

淋球菌nspA和大肠杆菌ltB融合基因的构建、表达及鉴定

通过基因工程的方法构建奈瑟氏淋球菌表面蛋白A(Neisseria gonorrhoeae surface protein A,nspA)和大肠杆菌不耐热肠毒素B亚单位(B subunit of Escherichia coli heat-labile enterotoxin,ltB)融合基因的原核表达载体,对其进行表达与鉴定,为后续融合蛋白LTB-NspA的生物活性分析及其作为淋球菌粘膜免疫疫苗的研究奠定基础.用PCR法从标准菌株分别扩增出nspA、ltB基因,用重组PCR法通过接头将ltB与nspA融合,将其插入pET-30a中,转入BL21中表达.经测序、SDS-PAGE和Western blot分析,证实成功构建了1tB-nspA融合基因的原核表达载体,并在BL21中表达.ltB-nspA融合基因的成功表达,为进一步研究其生物活性及淋球菌粘膜免疫疫苗的研究奠定了一定基础.     

Carboxyl-terminal domain of human apolipoprotein E: expression, purification, and crystallization.

Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.     

Gene cloning, expression, and characterization of a novel beta-mannanase from Bacillus circulans CGMCC 1416

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-beta-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa beta-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58 degrees C and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.     

Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli

Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.     

Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli

A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E. coli cell. The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P. Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography.     

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure.This structure causes highly efficient fusion protein folding.     

Expression of bovine inhibin beta subunit in Escherichia coli.

1. A DNA fragment encoding the beta subunit of bovine inhibin was amplified using the polymerase chain reaction and was cloned in plasmids pUC8 and pUR291. 2. Cultures of Escherichia coli TG2 harbouring pKDK37, a pUR291-derived recombinant plasmid, produced a novel protein with a molecular weight of 130,000 corresponding to a beta-galactosidase-inhibin beta fusion protein. 3. The fusion protein was purified from inclusion bodies by solubilization in 8 M urea followed by an ion-exchange and gel permeation chromatography. 4. Analysis by immunoblotting and competitive radioimmuno assay revealed that the fusion protein was recognized by a monoclonal antibody raised against a chemically synthesized peptide for amino acid residues from +82 to +114 of the beta subunit of the bovine inhibin thereby confirming its identity.