Enhancing recombinant protein yields in Escherichia coli using the T7 system under the control of heat inducible lambda PL promoter

A recombinant plasmid containing the complete lacZ gene downstream of the T7 promoter was used to transform Escherichia coli containing another plasmid which had the T7 RNA polymerase gene under the control of heat inducible lambda PL promoter. This recombinant E. coli containing the two plasmids was studied in order to enhance beta-galactosidase expression. The heat shock time which effectively regulates the T7 RNA polymerase was optimized and best expression of beta-galactosidase was obtained with 2 min heat shock. Substrate feeding increased the duration of log phase and allowed induction at a higher cell density without affecting the specific activity. A high cell density (7 g l-1) and high specific activity (approximately 20,000 U) were achieved which effectively increased the product concentration 18-fold.     

Expression and secretion of interferon-alpha 1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene

A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.     

Efficient solubilization of inclusion bodies

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.     

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.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

Refolding and structural characteristic of TRAIL/Apo2L inclusion bodies from different specific growth rates of recombinant Escherichia coli

TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) was produced mainly as inclusion bodies (IBs) by recombinant Escherichia coli with a temperature-inducible expression system. The yield of TRAIL type 2 IBs at higher preinduction specific growth rate (mu = 0.15 h-1) was higher than that of TRAIL type 1 IBs at lower preinduction specific growth rate (mu = 0.05 h-1). With the same optimized refolding protocols, two types of IBs exhibited different refolding features. Refolded type 1 IBs had higher recovery of more than 80% compared with type 2 IBs (57-63%). By the measurements of fluorescence and CD spectroscopy, type 1 TRAIL IBs dissolved by urea appeared to be a closer secondary structure to the native TRAIL than type 2. Furthermore, with trypsin treatment, the striking decrease in stability of type 1 IBs against protease digestion cannot be attributed to their small size particles observed by scanning electron microscope and probably depend on different protein structure properties between the two IBs. Different properties of inclusion bodies were mainly influenced by different physiological states of the cells just prior to the induction.     

新型瞬时报告载体pRSET-EGFP的构建及表达

目的：利用分子生物学技术和方法将pRSET-B质粒改建为带有绿色荧光蛋白(GFP)突变体基因(GFP-S65T)的新型瞬时表达载体pRSET-EGFP,并在E．coli．BL21中得到GFP基因的高效表达。方法：PCR法从pEGFP质粒克隆GFP-S65T cDNA并在5’末端引入KpnⅠ的位点。将扩增出来的GFP-S65T基因和pRSEY-B质粒用HindⅢ和KpnⅠ双酶切后连接构成重组质粒。用化学法把重组质粒转化到E．coli．BL21中,培养发酵液。OD540=0．4时加入IPTG诱导GFP-S65T基因转录和表达,合成绿色荧光蛋白。还对诱导条件进行了优化,发酵液OD540=0．4加入IPTG可以得到最优表达。结果：通过Ni^2 柱亲和层析,纯化得到绿色荧光蛋白,SDS-PAGE电泳检测,分子量为27kDa,与文献报道值一致。这说明Ni^2 柱能够有效的纯化表达产物。结论：成功构建了新型瞬时表达载体pRSET-EGFP,并且在E．coli．BL21中得到高效表达。     

Molecular amplification and purification of the E. coli recC gene product.

The level of recC gene expression has been analysed using Mud(bla lac) fusions to the recC promoter. The constitutive level of expression is very low and remains so even under SOS inducing conditions. The recC gene product has been amplified by harnessing the gene to the phage lambda leftward promoter in a plasmid. From cells harbouring this plasmid, RecC protein, which represented approximately 6% of the total cellular protein, was purified to electrophoretic homogeneity.     

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。     

trans activation of type 1 interferon promoters by simian virus 40 T antigen.

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.     

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.     

GNA成熟蛋白基因亚克隆及其原核表达载体构建

雪花莲外源凝集素（GNA）对刺吸式昆虫和某些咀嚼式昆虫以及多种线虫均有毒性,从含GNA前体蛋白基因的质粒中亚克隆出GNA的成熟蛋白基因MGNA,将MGNA基因插入大肠杆菌表达载体pET22b的不同位点,再经测序验证,得到了三种不同表达形式的GNA原核表达载体;22bG1(分泌型融合GNA蛋白）,22bG2(包涵体型GNA蛋白）,22bG3(分泌型天然GNA蛋白）,这为进一步在大肠杆菌中表达GNA和将GNA制成生物农药奠定了基础。     

Fostriecin产生菌Streptomyces pulveraceus遗传转化体系的建立与优化

【目的】建立并优化链霉菌Fostriecin产生菌Streptomyces pulveraceus的遗传转化系统。【方法】以整合型质粒pSET152为出发质粒,通过供体菌E.coli ET12567/pUZ8002与受体菌Streptomyces pulveraceus进行接合转移。【结果】确定了链霉菌Streptomyces pulveraceus的最佳接合转移条件:培养基为终浓度含15%甘氨酸的MS培养基;孢子热激条件为50°C 10 min;阿伯拉霉素覆盖的时间为18 h,终浓度为20 mg/L。同时,把组成型启动子ermE+与绿色荧光蛋白基因(gfp)克隆到pSET152载体上,通过接合转移整合到该链霉菌中,gfp获得表达。【结论】建立Fostriecin产生菌的遗传转化系统,并发现甘氨酸能显著提高链霉菌的接合转移效率。     

Reduction of N-terminal methionylation while increasing titer by lowering metabolic and protein production rates in E. coli auto-induced fed-batch fermentation

A standard fed-batch fermentation process using 1 mM isopropyl-β-D: -thiogalactopyranoside (IPTG) induction at 37 °C in complex batch and feed media had been developed for manufacturing of a therapeutic protein (TP) expressed in inclusion bodies (IBs) by E. coli BL21 (DE3) driven by T7 promoter. Six unauthentic TP N-terminal variants were identified, of which methionylated TP (Met-TP) ratio was predominant. We hypothesized that lowering metabolic and protein production rates would reduce the Met-TP ratio while improving TP titer. The standard process was surprisingly auto-induced without added IPTG due to galactose in the complex media. Without changing either the clone or the batch medium, a new process was developed using lower feed rates and auto-induction at 29 °C after glucose depletion while increasing induction duration. In comparison to the standard process, the new process reduced the unauthentic Met-TP ratio from 23.6 to 9.6 %, increased the TP titer by 85 %, and the specific production yield from 210 to 330 mg TP per gram of dry cell weight. Furthermore, the TP recovery yield in the purified IBs was improved by ~20 %. Adding together, ~105 % more TP recovered in the purified IBs from per liter of fermentation broth for the new process than the standard process. The basic principles of lowering metabolic and production rates should be applicable to other recombinant protein production in IBs by fed-batch fermentations.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Gene 26 of bacteriophage T4 basal plate. I. Two expression products of the cloned gene

We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter. The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression. We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed. Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26. The 8-9 kDa protein was shown to be expressed from the end region of gene 26. Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame.