A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.     

含Arg9的人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达和内化作用的研究

目的：构建、表达和纯化带有转膜结构域Arg9的ScFv14/EGFP融合蛋白, 并对纯化产物的亲和 活性和内化作用进行研究。 方法： 将Arg9的编码序列分别重组到ScFv14/EGFP基因的5'端或3'端或二者之间,将它们分别克隆入原核表达载体pET32a,转化大肠杆菌BL21(DE3)LysS进行诱导表达和纯化,用间接ELISA方法检测表达产物与HBsAg的亲和活性,并用间接免疫荧光检测纯化蛋白的内化活性。 结果：经测序及酶切鉴定证实四种融合基因序列完全正确.SDS-PAGE和Western blot证实四种融合基因成功表达和纯化.间接ELISA检测证实四种融合蛋白均具有HBsAg结合活性,间接免疫荧光检测显示N端带有Arg9的融合蛋白有较强的内化作用,而且不内化进入HBsAg非表达的细胞。 结论：成功构建、表达和纯化了ScFv14/EGFP融合蛋白和三种带有Arg9的ScFv14/EGFP融合蛋白,纯化产物均具有与抗原HBsAg亲和的活性,N端带有Arg9的融合蛋白与靶细胞作用有较强的内化作用。     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

霍乱毒素B亚基融合蛋白表达系统的构建

霍乱毒素Ｂ亚基（ＣＴＢ）是良好的免疫佐剂和载体蛋白。本研究通过定点突变,在ＣＴＢ基因（ｃｔｘＢ）３′端终止密码前引入了限制性内切酶ＥｃｏＲＩ,构建了质粒ｐＭＣ０５。ｐＭＣ０５中ＣＴＢ与下游ｌａｃＺ′基因阅读框架相同,转化大肠杆菌后能够表达ＣＴＢ与β－半乳糖苷酶α肽的融合蛋白;所表达的融合蛋白能与ＧＭ１结合,说明融合蛋白保持ＣＴＢ的基本高级结构和生物学活性;融合蛋白能与抗－ＣＴＢ抗体结合,说明融合蛋白具有ＣＴＢ的抗原性。以上结果表明：通过将外源抗原决定簇基因融合至ｃｔｘＢ的３′端,在大肠杆菌中表达融合蛋白,构建基因工程肽苗是可行的。还探索了转录终止序列对融合基因蛋白表达水平的影响,构建了高效表达融合蛋白的载体－宿主系统。     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Immunological characterization of the Campylobacter jejuni 72Dz/92 cjaD gene product and its fusion with B subunit of E. coli LT toxin

Campylobacter jejuni 72Dz/92 cjaD gene, orthologue of C. jejuni NCTC 11168 cj0113, C. jejuni M275 omp18 and C. jejuni ATCC 29428 omp18, has been cloned, sequenced and analysed from the viewpoint of its immunological attributes. Neither the 5' nor 3' fragment of the cjaD encodes protein capable of reacting with anti-Campylobacter antibodies. Several fusions of the cjaD with eltB, which encodes B subunit of the E. coli LT toxin, have been constructed. The hybrid proteins, which differ in respect to their cellular localization, retain the ability to react with GM1 and are recognized by the antibodies specific for both moieties of the proteins. The fusion protein equipped with signal sequence, reveals a stronger affinity to GM1 than its equivalent which is unable to cross the inner membrane. Two recombinant plasmids (pUWM405 expressing both LTB and CjaD proteins and pUWM299 containing cjaD gene fused into 3' end of Escherichia coli eltB gene lacking signal sequence) were introduced into avirulent Salmonella enterica serovar Typhimurium strain where they are stably maintained.     

Production of recombinant peptides as fusions with SUMO

Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.     

Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions.

Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway. The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced. E. coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence. Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA). Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane. The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides.     

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.     

Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli.

Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter. The over-expressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.     

Isolation and characterization of human HSP70 expressed in Escherichia coli

A plasmid containing the human HSP70 gene was used to transfect and express the protein in Escherichia coli. The bacterial product was a fusion protein containing 640 amino acids of HSP70, plus 33 additional NH2 terminal amino acids; 12 from the bacterial expression vector and 21 from a 5' human sequence that is not normally translated. It was partially purified by ion-exchange and ATP-Sepharose affinity column chromatography. The bacterially produced human HSP70 protein was then compared with HSP70 obtained from cultured 293 cells. Both shared the same staphylococcal V8 protease peptide fragment pattern, ATP binding, and a weak ATPase activity (about 10-15 nmol ATP hydrolyzed per milligram protein per minute at 30 degrees C). The bacterially produced human HSP70 protein differed in its V8 protease pattern with an E. coli ATP-binding protein that corresponded in molecular mass to the E. coli dnaK gene product. Mutants in the human HSP70 gene were constructed which significantly reduced a predicted major alpha-helical domain in the HSP70 molecule that has partial homology to an ATP-binding site of several protein kinases. One HSP70 mutant clone contained a deletion of 20% at the NH2 terminus, and expressed a 57-kDa product, while the other was missing the middle 50% of the gene (40-kDa product). Neither protein fragment bound to an ATP affinity column, suggesting that ATP binding to HSP70 may be conformationally affected by a region about 20% internal to the NH2 terminal end of the molecule. Recently, a similar location of the ATP-binding site has been reported by Milarski and Morimoto (27).     

中华眼镜蛇短链神经毒素cDNA的克隆及在大肠杆菌中的表达

利用RT－PCR技术从中华眼镜蛇毒腺组织中成功地克隆了短链神经毒素CDNA。测序结果表明,该基因开放阅读框架编码83个氨基酸残基,其中对个为信号肽,成熟肽为62个氨基酸残基。该基因与GenBank报道的相同物种的神经毒素基因有相当的同源性,不同物种之间的信号肽序列十分保守。将短链神经毒素CDNA再经PCR扩增除去信号肽序列,克隆到pT7ZZ表达质粒中,转化E.coliBL21（DE3）后,经IPTG诱导可高效表达分子量为23kDa②左右的融合蛋白。表达产物占菌体总蛋白的25％左右。     

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.     

Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level

Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme.     

Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein.     

Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein

Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase.