Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.

A 454 base pair fragment of double stranded DNA consisting of a gene for a human immune interferon (hIFN-gamma), initiation and termination signals plus appropriate restriction endonuclease sites, was totally synthesized. The synthesis involved preparation of 62 oligodeoxyribonucleotides by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. This synthetic gene was expressed in E. coli under the control of the lac UV5 promoter. The product has antiviral activity which was acid labile and completely neutralized by antiserum to hIFN-gamma but not by antiserum to hIFN-alpha or hIFN-beta. Molecular weight of hIFN-gamma produced by E. coli was estimated to be about 32,000 and 17,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively.     

Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.     

Stabilisation of human interferon beta synthesis in Escherichia coli by zinc ions

Human interferon beta synthesized in Escherichia coli is unstable and toxic for the bacterial cell. Zinc ions are able to stabilise interferon beta in E. coli probably by inhibiting the action of cell internal proteinase(s) which affect the half-life of this foreign protein. As a result up to one order of magnitude more active IFN-beta can be detected in Zn2+-treated E. coli cells.     

Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.     

The regulatory elements of araBAD operon,contrary to lac‐based expression systems,afford hypersynthesis of murine,and human interferons in Escherichia coli

The overexpression of four different interferons, i.e., murine interferon α1 and human interferons α1, α8, and α21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon α1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining β‐galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

猪α1-干扰素的基因改造与高效原核表达

poIFNα1基因中含有大量的大肠杆菌稀有密码子,为了获得高表达,使用了大肠杆菌的偏爱密码子,人工合成了poIFN|α1成熟蛋白编码基因。在保留编码蛋白序列的同时,使其5′端A+T的含量增加到最大限度,并将其终止密码子改为TAA。将合成的poIFNα1成熟蛋白编码基因插入原核单纯表达载体pRLC中,转化大肠杆菌DH5α。实现了poIFNα1在大肠杆菌中的高效表达,表达产物以包涵体形式存在。纯化的包涵体经含DTT的6 mol/L盐酸胍的变性液溶解及含GSHGSSG的复性液复性处理,复性后的表达产物浓缩后经凝胶层析纯化,细胞病变抑制法测定表明,重组poIFNα1具有较高的抗病毒活性,约为6.4x10⁶u/mg。      

Synthesis of human fibroblast interferon by E. coli

A cDNA library was constructed using mRNA from human fibroblasts induced with poly(I):poly(C). A bacterial clone containing fibroblast interferon cDNA sequences was identified by hybridization to a cDNA probe synthesized using deoxyoligonucleotide primers which hybridize to fibroblast interferon mRNA specifically. Expression plasmids were constructed which permitted the synthesis in E. coli of 8 x 10(7) units of human fibroblast interferon per liter of culture. The bacterially produced fibroblast interferon is indistinguishable from authentic human fibroblast interferon by several criteria.     

重组人干扰素α-2b在大肠杆菌中分泌表达

人干扰素α-2b原始基因在重组原核工程菌中表达量偏低,所以我们在不改变干扰素原有氨基酸组成的前提下,根据大肠杆菌密码子偏爱性使用定向突变技术对huIFNα-2b基因进行点突变。将大肠杆菌STⅡ信号肽基因与突变后huIFNα-2b基因融合并于信号肽5′端和huIFNα-2b基因3′端引入合适的酶切位点。融合基因克隆至载体pCSE,pET-22b和pPAK4L中,此3种载体分别含有组成型启动子、T7启动子和phoA启动子。融合基因在载体pCSE中表达量很低,其中约有50%的目标蛋白能够成功实现分泌。在E.coliBL21中,pET-22b经过IPTG诱导可以实现huIFNα-2b的高表达,但STⅡ信号肽不能被有效切除。含有phoA启动子的载体pPAK4L其在E.coliW3110中可以实现huIFNα-2b较高水平的分泌表达,经过低磷诱导其表达量最高可至20μg/mL(A550)菌液,约有30%的目标蛋白质信号肽能够被成功切除并分泌到胞间质中。     

Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.     

Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium.

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.     

Bacterial synthesis of a novel human leukocyte interferon.

A novel human leukocyte interferon cDNA clone (LeIF B) was identified in a cDNA library prepared using polyadenylated mRNA of a myeloblastoid cell line. The nucleotide sequence of LeIF B differs significantly from other published leukocyte interferon cDNA sequences. An expression plasmid was constructed which directs the synthesis in E. coli of 8 x 10(7) interferon units per liter of culture. LeIF B exhibits markedly different specificities from another bacterially synthesized human leukocyte interferon, LeIF A.     

Translational features of human alpha 2b interferon production in Escherichia coli

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.     

Expression of the chemically synthesized coding region for the cytotoxin alpha-sarcin in Escherichia coli using a secretion cloning vector

The coding region for the cytotoxin alpha-sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides. An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-OmpA2. The enzyme encoded by the chemically synthesized gene expressed in E. coli displayed properties identical to those of native alpha-sarcin isolated from A. giganteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays.     

Creation of artificial hybrid operons with partially overlapping genes to achieve an expression of heterologous genes in Escherichia coli cells

A new method of optimization of foreign gene expression in E. coli, based on the construction of hybrid operons with partially overlapping genes is described. The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis. Such an approach has provided the synthesis of human interferon alpha F in E. coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture. The reinitiation of the distal gene translation is shown to take place in the intercistronic region. Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter. The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E. coli trpE-trpD genes are used for translational coupling.     

Recombinant human leukocyte interferon produced in bacteria has antiproliferative activity

Recombinant human leukocyte interferon synthesized by Escherichia coli possesses antiproliferative activity in addition to antiviral activity. When the ability to inhibit multiplication of lymphoblastoid Daudi cells was examined, the growth-inhibitory capacity of recombinant leukocyte interferon was equivalent to that exhibited by crude human leukocyte interferon or by the homogeneous gamma 2 species of leukocyte interferon synthesized by human cells.