外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

The hha gene modulates haemolysin expression in Escherichia coli

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202-312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.     

大鼠OB基因克隆及其在大肠杆菌中的表达

采用ＲＴＰＣＲ技术扩增大鼠ＯＢｃＤＮＡ编码区序列。ＰＣＲ产物酶切后定向克隆至ｐＵＣ１９质粒。经核苷酸序列测定表明与文献报道的大鼠ＯＢｃＤＮＡ编码区序列一致。继之构建了ｐＢＶ２２０ｒＯＢ表达质粒并获得了ＯＢ基因在大肠杆菌中的特异表达。大鼠ＯＢ基因产物的获得为研究肥胖与某些非传染性疾病（如糖尿病、高血压病）间的关系提供了条件     

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.     

Global gene expression in Escherichia coli biofilms

It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared with planktonic growth. Genes encoding proteins involved in adhesion (type 1 fimbriae) and, in particular, autoaggregation (Antigen 43) were highly expressed in the adhered population in a manner that is consistent with current models of sessile community development. Several novel gene clusters were induced upon the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces.     

Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.     

鱼腥藻II型果糖-1,6-二磷酸醛缩酶基因的克隆及其在大肠杆菌中的高效表达

随着更多蓝藻全基因组序列测定完成,蓝藻基因工程研究现已进入后基因组时代。2001年Kaneko等人完成了鱼腥藻7120全基因组序列测定,随后人们利用生物信息学的方法对其中一些基因的功能进行了预测,包括II型果糖-1,6-二磷酸醛缩酶(FBA)基因,但该基因是否编码II型果糖-1,6-二磷酸醛缩酶及该产物的相关酶学特性至今尚未见报道。本文通过PCR克隆到鱼腥藻7120中预测的II型FBA基因,插入到质粒pET-32a的相应位点,构建成原核表达载体pET-FBA-II。蛋白电泳结果显示,重组蛋白的表达量占总蛋白含量的23.4%,与蛋白分子标记相比,其分子量约为40 kDa。酶学活性测定结果表明,其蛋白粗提物的比活力为11.8 U (mg protein)-1,具有标准II型FBA活性。本研究不仅证实了Cyanobase中关于该基因功能的预测,也为进一步研究该基因表达产物的生理生化特性及功能提供了重要条件。     

山梨糖脱氢酶基因在大肠杆菌染色体上整合及表达

以质粒pKF3为模板,扩增出两翼与ptsG基因上下游序列同源,中间为氯霉素抗性基因的DNA片段,连至pMD18T载体,构建得到pMD18PC。以质粒pQE60SDH为模板,扩增山梨糖脱氢酶基因sdh,与pMD18PC连接,得到pMD18PCSDH。将其用PvuⅡ酶切,回收含ptsG1catsdhptsG2的目的片段,电转化至JM109/pKD46,在Red重组酶的作用下,外源DNA片段与染色体上对应区域发生同源重组,将基因ptsG敲除,替换为catsdh基因,获得整合sdh基因的JM109s。经检测JM109s具有山梨糖脱氢酶活性。以 ptsG基因上下游序列为引物,JM109s基因组DNA为模板进行PCR,扩增产物测序结果表明sdh基因染色体整合成功。     

Direct expression of urogastrone gene in Escherichia coli

Human epidermal growth factor (urogastrone; UG) is a 53-amino acid polypeptide hormone. A 192-bp DNA fragment containing the coding sequence for methionyl UG (Met-UG) and the ribosome-binding site (RBS) was chemically synthesized and placed downstream from the promotor for the Escherichia coli outer-membrane lipoprotein gene (lpp) on a plasmid. E. coli cells harboring the plasmid directed the synthesis of Met-UG at 10(2)-10(3) molecules per cell. Next, the coding sequence for Met-UG was inserted in a runaway-replication plasmid and expressed under the control of the lpp promoter and the RBS derived from bacteriophage Mu cII gene. Upon heat induction, the cells harboring the recombinant plasmid synthesized 10(5) molecules of Met-UG per cell.     

Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the ? 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.     

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.     

Chemical synthesis of a human interferon-alpha 2 gene and its expression in Escherichia coli

A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.     

Processivity errors of gene expression in Escherichia coli

Not all ribosomes that initiate translation of an mRNA sequence will successfully complete it and produce a full-length protein product. By comparing the amounts of lacZ monomer and lacZ dimer protein expressed from a plasmid in a strictly controlled assay, we calculate a dimer to monomer ratio of 0.76. We interpret this to mean that ribosomes have a 76% chance of completing the synthesis of a beta-galactosidase polypeptide. The remaining 24% of the initiated chains end in processivity accidents. For the wild-type, premature RNA polymerase termination is found to account for roughly one-third of the processivity accidents. For the hyperaccurate SmP mutant, we observe a processivity of 0.28, but the presence of streptomycin improves this to 0.50. Thus, the hyperaccuracy with respect to missense substitutions for this mutant is accompanied by a reduced processivity. Addition of streptomycin increase the first error class and reduces the second one. This finding is relevant to the optimization of ribosome function and the growth performance of ribosome mutants.     

Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli

The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.     

Chemical synthesis of a human fibroblast interferon gene and its expression in Escherichia coli

Using the solid phase phosphotriester method, a gene coding for human fibroblast interferon consisting of 166 amino acid residues was chemically synthesized. The gene obtained by ligation of 61 synthesized oligodeoxyribonucleotide fragments was inserted into the downstream of tryptophan promoter, and expressed in E. coli. Lysate of this E. coli showed the antiviral activity which was specifically neutralized by anti-fibroblast interferon antibody. No particular advantage was observed in the expression efficiency by the synthetic gene over that by the native gene.