High level expression of porcine growth hormone in Escherichia coli from an expression vector containing bacteriophage lambda PL and N gene untranslated region

An Escherichia coli expression vector, pG408N containing a PL promoter and the upstream untranslated region of the N gene of bacteriophage lambda has been constructed. We have designed a PvuII site immediately behind the untranslated region. A DNA fragment starting with an initiation codon ATG could be inserted into this site for expression. This vector also contains 7 additional cloning sites downstream from the PvuII site. A gene could be cloned into one of these sites and the 5' sequence of this gene could be modified with synthetic oligonucleotides and ligated to the PvuII for the purpose of increasing gene expression. We have also cloned the lambda cl gene into a p15A plasmid. Cotransformation of this plasmid with the expression vector allows the cloning vector pG408N to be used in any E. coli strain. Using this system, we were able to express porcine growth hormone to approximately 35% of total proteins in E. coli DH5 alpha.     

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid vector for overproduction and export of recombinant protein in Escherichia coli: efficient one-step purification of a recombinant antigen from Echinococcus multilocularis (Cestoda)

We describe the use of the Escherichia coli plasmid vector, pVB2, for high-level expression and export of recombinant protein. The pBR322 derivative pVB2 harbors the mglB gene, which codes for the galactose-binding protein (GBP) of E. coli. GBP is exported into the periplasmic space of the bacterial cell. Gene mglB contains an EcoRI restriction site close to its 3' end which allows simple in-frame insertion of EcoRI fragments obtained from recombinant lambda gt11 phages. The pVB2 vector was used to express an antigen from Echinococcus multilocularis. The recombinant protein amounted to over 50% of total cellular protein and could be efficiently isolated from the periplasm by osmotic shock. The application of the purified antigen in an ELISA enabled a clear and specific detection of anti-Ec. multilocularis antibodies in human patients' sera, which had been immunosorbed with a periplasmic extract (containing wt GBP) before investigation. These data show the general usefulness of pVB2 as an expression vector for producing in E. coli diagnostically relevant antigens from any infective organism.     

Yeast DNA topoisomerase II is encoded by a single-copy, essential gene

The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11. The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (S1 nuclease mapping) for the 5' end and mapping of the polyA tail portion of a cDNA fragment for the 3' end. The predicted size of the message agrees with the length of the message as determined by Northern blot hybridization analysis. The identity of the gene was confirmed by expressing the gene in E. coli from the E. coli promoter lac UV5 to give catalytically active yeast DNA topoisomerase II. Disruption of one copy of the gene in a diploid yeast creates a recessive lethal mutation, indicating that the single DNA topoisomerase II gene of yeast has an essential function.     

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.     

A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine pancreatic phospholipase A2

A novel expression plasmid (pTO-N) has been constructed that allows for the production of large quantities of foreign proteins (or fragments thereof) in an unfused state. The vector has a strong and tightly regulated T7 gene 10 promoter and the ompA Shine-Dalgarno (SD) sequence, followed by the ompA sequence and a cloning linker region. The mRNAs produced by the vector are protected by secondary structures at both ends of the mRNAs. The OmpA signal peptide directed the synthesized proteins into the periplasmic space of Escherichia coli. Phospholipase A2 and prophospholipase A2 from bovine pancreas have been produced to a high level by using this expression vector. One additional feature, which is essential for the stable maintenance of the plasmid in the E. coli expression host, BL21 (DE3)[pLysS], is the shortened distance between the 5' secondary structure sequence (immediately following the gene 10 promoter) and the SD sequence. This vector could be particularly useful for synthesis of toxins in E. coli.     

Protein purification by ultrafiltration using a beta-galactosidase fusion tag

The use of beta-galactosidase (465 kDa) as a fusion tag for ultrafiltration-based protein purification has been investigated. The target protein studied was thermophilic glucose dehydrogenase (157 kDa, GDH) from Thermoplasma acidophilum. An expression vector was constructed comprising the lacZ gene fused to a factor Xa cleavage sequence that was attached to the 5' end of the GDH gene. This gene fusion was expressed in Escherichia coli JM109 to yield a soluble protein that exhibited activities for both enzymes. Cleavage of this fusion protein (622 kDa) by factor Xa gave two smaller proteins that showed individual beta-galactosidase and GDH activity. A two-stage diafiltration process for protein purification was used in an ultrafiltration stirred cell. In the first stage, a 500 kDa membrane was used to retain the fusion protein and transmit smaller E. coli host proteins. Approximately 80% of the GDH activity was retained in this step. Following cleavage, the second stage utilized a 300 kDa membrane to fractionate the beta-galactosidase and GDH. No beta-galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate.     

Molecular cloning, sequencing, and mapping of the bacteriophage T2 dam gene.

Bacteriophage T2 codes for a DNA-(adenine-N6)methyltransferase (Dam), which is able to methylate both cytosine- and hydroxymethylcytosine-containing DNAs to a greater extent than the corresponding methyltransferase encoded by bacteriophage T4. We have cloned and sequenced the T2 dam gene and compared it with the T4 dam gene. In the Dam coding region, there are 22 nucleotide differences, 4 of which result in three coding differences (2 are in the same codon). Two of the amino acid alterations are located in a region of homology that is shared by T2 and T4 Dam, Escherichia coli Dam, and the modification enzyme of Streptococcus pneumoniae, all of which methylate the sequence 5' GATC 3'. The T2 dam and T4 dam promoters are not identical and appear to have slightly different efficiencies; when fused to the E. coli lacZ gene, the T4 promoter produces about twofold more beta-galactosidase activity than does the T2 promoter. In our first attempt to isolate T2 dam, a truncated gene was cloned on a 1.67-kilobase XbaI fragment. This construct produces a chimeric protein composed of the first 163 amino acids of T2 Dam followed by 83 amino acids coded by the pUC18 vector. Surprisingly, the chimera has Dam activity, but only on cytosine-containing DNA. Genetic and physical analyses place the T2 dam gene at the same respective map location as the T4 dam gene. However, relative to T4, T2 contains an insertion of 536 base pairs 5' to the dam gene. Southern blot hybridization and computer analysis failed to reveal any homology between this insert and either T4 or E. coli DNA.     

A new vector for cloning of genes and replicons in yeasts

A novel Escherichia coli-Saccharomyces cerevisiae shuttle vector lambda MAN78 has been constructed. The vector contains phage lambda 47.1 DNA, Sacch. cerevisiae chromosomal segment with TRP1 gene and the yeast ARS1 replicator. This vector may be propagated as a phage and, similar to parental lambda 47.1, allows direct selection of large DNA inserts (15-24 kbp) in E. coli. lambda MAN78 can efficiently transform LiCl-treated yeast cells (3-5 X 10(3) transformants per 1 microgram DNA). Replication of hybrid molecules in E. coli cells does not influence the ability of the molecules to transform yeast cells and replicate in the cells.     

Cloning and expression in Escherichia coli of the TL-DNA gene 4 of Agrobacterium tumefaciens under the control of the PR promoter of bacteriophage lambda

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows: i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of beta-galactosidase. The hybrid gene was poorly expressed from the upstream lambda PL promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the lambda PR promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-beta-galactosidase was identified among the Lac+ clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried beta-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-beta-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the lambda PR promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.     

Expression of the E2 open reading frame of papilloma viruses BPV1 and HPV6b in Escherichia coli

A new expression vector, pRA10, has been constructed for the expression of the open reading frames (ORF) of bovine (B) and human (H) papilloma viruses (PV). This vector is a derivative of pAJ pi and contains 15 restriction sites proximal to the lambda PL promoter, offering considerable versatility for insertion of different ORFs. This vector was used specifically to express the E2 ORF gene products from BPV1 and HPV6b at high level in Escherichia coli. The genuine nature of these proteins was demonstrated by restriction map analysis of expression vector plasmids to insure proper orientation, nucleotide sequence analysis to demonstrate in-frame insertion, E2 ORF protein production by expression-vector plasmids, and not by appropriate controls and, immunoprecipitation of E2 proteins by antibody specific for a common N-terminal sequence derived from the expression vector.     

A system for dual protein expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

Direct role of the Escherichia coli Dam DNA methyltransferase in methylation-directed mismatch repair.

The T4 dam+ gene has been cloned (S. L. Schlagman and S. Hattman, Gene 22:139-156, 1983) and transferred into an Escherichia coli dam-host. In this host, the T4 Dam DNA methyltransferase methylates mainly, if not exclusively, the sequence 5'-GATC-3'; this sequence specificity is the same as that of the E. coli Dam enzyme. Expression of the cloned T4 dam+ gene suppresses almost all the phenotypic traits associated with E. coli dam mutants, with the exception of hypermutability. In wild-type hosts, 20- to 500-fold overproduction of the E. coli Dam methylase by plasmids containing the cloned E. coli dam+ gene results in a hypermutability phenotype (G.E. Herman and P. Modrich, J. Bacteriol. 145:644-646, 1981; M.G. Marinus, A. Poteete, and J.A. Arraj, Gene 28:123-125, 1984). In contrast, the same high level of T4 Dam methylase activity, produced by plasmids containing the cloned T4 dam+ gene, does not result in hypermutability. To account for these results we propose that the E. coli Dam methylase may be directly involved in the process of methylation-instructed mismatch repair and that the T4 Dam methylase is unable to substitute for the E. coli enzyme.     

Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein)

A new secretion vector, pEAP84 which contained a unique restriction site (BglII) at the 3' end of the penicillinase gene to produce a fused protein, and the Ex-kil region to make the outer membrane permeable, was constructed from pEAP82. A recombinant plasmid p84h06, which contained a synthetic gene for human calcitonin with a cyanogen bromide cleavage site at the junction site of the fused protein, was constructed and introduced into Escherichia coli. The hybrid protein produced in E. coli carrying p84h06 was secreted into the culture medium. The amino acid composition of this product was consistent with that deduced from the DNA sequence. Mature calcitonin was obtained following cyanogen bromide cleavage of the fused protein.     

具有抗HIV活性的突变型天花粉蛋白TCSYFF-KR的构建及原核表达

目的：构建具有抗HIV活性的突变型天花粉蛋白（TCS）,并将其在原核系统内进行表达与纯化。方法：借助计算机预测TCS分子上可能的抗原决定簇（YFF81-83和KR173-174）,并依此设计适当的突变引物;以栝楼基因组DNA为模板,利用重组PCR技术扩增双突变型TCS全长基因,经BamHI和EcoRI双酶切后与原核表达载体pRSET-A连接,转化感受态大肠杆菌DH5α,提取质粒进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21（DE3）,经IPTG诱导表达后,对表达产物进行Western印迹鉴定;用Ni-NTA亲和层析柱对所获突变型TCS蛋白进行纯化。结果：构建了突变型TCSYFY-KR,并获得了该蛋白在大肠杆菌内的可溶性高效表达;经Ni-NTA亲和层析柱纯化,产生大量均一的突变型TCS蛋白。结论：TCS的定点突变及其在原核系统内的表达,为基因工程方法改造TCS提供了一条新途径。     

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

Restriction site-free insertion of PCR products directionally into vectors

A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.