Purification and characterization of recombinant bovine growth hormone produced in Escherichia coli

Using the pUBJ10 plasmid containing the modified bovine growth hormone (bGH) cDNA, large production has been attempted in E. coli BL21 strain. The bGH was highly expressed upto the level of 35% of total cell proteins by IPTG induction and temperature shift to 40°C. The recombinant bGH (rbGH) was isolated from inclusion bodies by solubilization in 10 M urea and followed by DEAE-TOYOPEARL 650C ion exchange and Sephadex G-100 column chromatography. The pUBJ10-derived bGH was eluted at 25.28 min similar to the standard bGH (at 25.18 min) by reverse-phase HPLC. The analysis of N-terminal amino acid showed that the mature bGH has glutamic acid as a first amino acid in agreement with DNA sequencing data. The biological activity was indirectly measured by radioreceptor assay and compared with a pituitary-derived bGH.     

Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains

Recently lactose mediated auto-induction in Escherichia coli has gained a lot of interest because higher protein titer could be achieved without the need to monitor growth and add inducer at the proper time. In this study a high level therapeutic protein production by auto-induction was observed in E. coli BL21 using either T7 or tac promoters in the modified Luria Bertani (mLB) medium containing soy peptone instead of tryptone in Luria Bertani (LB) medium. Based on medium analysis and spiking experiments it was found that 0.4 mM galactose from the soy peptone caused the auto-induction. E. coli cultures induced by galactose can saturate at considerably higher density than cultures induced by IPTG. Galactose is not consumed by E. coli BL21. Finally it has been demonstrated that auto-induction can be effectively used in fed-batch fermentation for the industrial production of a therapeutic protein. The principle of galactose mediated auto-induction should be able to apply to high throughput microplates, shake flasks and fed-batch fermentors for clone screening and therapeutic protein expression in E. coli gal(-) strains such as most commonly used BL21.     

AN OPTIMIZED PROTOCOL FOR OVERPRODUCTION OF RECOMBINANT PROTEIN EXPRESSION IN Escherichia coli

The gram-negative bacterium Escherichia coli (E. coli) offers a means for rapid, high-yield, and economical production of recombinant proteins. Here, a protocol for optimization of parameters involved in bacterial expression conditions is described. L-Asparaginase (ASNase II) was chosen as a model protein for our experiments. ASNase II gene (ansB) was cloned into the pAED4 plasmid and transformed into E. coli BL21pLysS (DE3)-competent cells. It was assumed that high cell density and high copy number of recombinant plasmid in the bacteria host could result in very high production of the recombinant protein. Circumstances for the overproduction of recombinant ASNase II including cell growth conditions, isopropyl β-D-1-thiogalactopyranoside (IPTG) level, ampicillin (Amp) concentration before and during IPTG induction, and cell density were optimized. Regarding the final optimization, overexpression of ASNase II was assessed on a large scale in LB medium. Periplasmic ASNase II was extracted using an alkaline lysis method. The extracted protein was purified by one-step DEAE-Sepharose fast-flow chromatography. ASNase II activity was considered an index for the protein expression. Applying the optimized practical protocol, protein production was significantly enhanced in comparison to the traditional IPTG induction method in the absence of a fermentor and can be applied for overexpression of other recombinant proteins.     

产姜黄素大肠杆菌工程菌的构建

姜黄素是姜科植物的特征性成分,具有重要的药理活性.文中利用姜黄素生物合成关键酶β-酮酰辅酶A合酶(Diketide-CoA synthase,DCS)基因和姜黄素合酶(Curcumin synthase,CURS)基因构建非天然融合基因DCS::CURS,并将其与4-香豆酰辅酶A连接酶(4-coumarate coen...     

N-糖酰胺酶F在大肠杆菌中的高效表达及其脱糖基化作用研究

从脑膜炎脓杆菌（Flavobacterium meningosepticum）基因组中通过PCR扩增了N-糖酰胺酶F（PNGase F）基因,经酶切后与表达载体pET28a连接,获得的重组质粒转入大肠杆菌BL21(DE3)。重组大肠杆菌经诱导表达和纯化提取后,获取大量高纯度N-糖酰胺酶F,其纯度达90%以上。试验证明,经纯化的重组N-糖酰胺酶F可以切除核糖核酸酶B、转铁蛋白和人IgG等糖蛋白上的N-糖链,具有脱糖基化作用。     

Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology

In this work, SVP2 from Salinivibrio proteolyticus strain AF-2004, a zinc metalloprotease with suitable biotechnological applications, was cloned for expression at high levels in Escherichia coli with the intention of changing culture conditions to generate a stable extracellular enzyme extract. The complete ORF of SVP2 gene was heterologously expressed in E. coli BL21 (DE3) by using pQE-80L expression vector system. In initial step, the effect of seven factors include: incubation temperature, peptone and yeast extract concentration, cell density (OD600) before induction, inducer (IPTG) concentration, induction time, and Ca(2+) ion concentrations on extracellular recombinant SVP2 expression and stability were investigated. The primary results revealed that the IPTG concentration, Ca(2+) ion concentration and induction time are the most important effectors on protease secretion by recombinant E. coli BL21. Central composite design experiment in the following showed that the maximum protease activity (522 U/ml) was achieved in 0.0089 mM IPTG for 24h at 30 °C, an OD600 of 2, 0.5% of peptone and yeast extract, and a Ca(2+) ion concentration of 1.3 mM. The results exhibited that the minimum level of IPTG concentration along with high cell density and medium level of Ca(2+) with prolonged induction time provided the best culture condition for maximum extracellular production of heterologous protease SVP2 in E. coli expression system.     

白喉毒素DAB389-（Gly4Ser）2-α-促黑激素工程菌发酵条件的初步研究

目的：为了提高融合蛋白白喉毒素DAB389-（Gly4Ser）2-α-促黑激素的表达量,研究工程菌发酵条件的最优化。方法：利用三角瓶模拟小剂量发酵,在不同的受体菌、培养基、培养温度、时间、pH值、诱导剂浓度、诱导时间和温度等因素变化下研究目的蛋白的表达量。结果：工程菌优化的发酵条件为：最佳表达菌种为大肠杆菌BL21（λDE3）,最适宜的培养基为M982,适合的pH值为7．0-7．4,最佳诱导温度为30℃,诱导时间为6h,诱导剂IPTG的最佳用量为1．4mmol/L。结论：获得了DAB389-（Gly4Ser）2-α-促黑素细胞激素工程菌的最优化发酵条件,为中试放大提供了理论依据。     

HER2胞外段的克隆与原核表达

应用RT-PCR技术从人乳腺癌细胞系SK-BR-3中克隆出人表皮生长因子受体2(human epidermal growth factorreceptor 2,HER2)基因的胞外段,并插入到表达载体pET-30a中,得到重组表达载体pET30-HER2(Ex)。将该载体转化至大肠杆菌BL21(DE3)细胞中,加入IPTG进行诱导表达,成功获得HER2胞外段蛋白。分别提取培养液上清、大肠杆菌周质腔、细胞质可溶性及不可溶性组分蛋白进行SDS-PAGE电泳分析,确定目的蛋白定位于大肠杆菌细胞质包涵体中。通过改变诱导温度、诱导物浓度、诱导起始菌体密度和诱导时间,寻找最佳表达条件,使目的蛋白的表达量达到最高。结果表明,在37℃下,OD600达到1.0时,经终浓度为0.1 mmol/L的IPTG诱导4 h,目的蛋白的表达量最高。将重组表达菌进行超声破碎,分离出包涵体组分,经Ni2+亲和层析纯化后获得了纯度>90%的HER2胞外段蛋白,从而为抗HER2抗体的制备及肿瘤疫苗的研究奠定了基础。     

Synthesis and excretion of alpha-amylase in vgb+ and vgb- recombinant Escherichia coli: a comparative study

Synthesis and excretion of alpha-amylase is investigated in batch cultures of Escherichia coli JM103[pMK57] (vgb-) and E. coli JM103[pMK79] (vgb+). While total production and excretion of alpha-amylase were promoted in Luria broth (LB) (excretion being as high as 87%), cell-mass-specific production of the enzyme was promoted in M9 in bioreactor cultures and in LB in shake flask cultures. Low aeration and agitation rates and presence of starch were conducive to alpha-amylase synthesis in E. coli JM103[pMK79]. Two-stage bioreactor operating strategies that will improve alpha-amylase production are proposed. The potential of these strategies is demonstrated via two-stage shake flask cultures.     

Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis

Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species. A region of the S. avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli. Production of the brown pigment was attained in E. coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG. The cloned S. avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E. coli vector. The gene involved in pigment production could be used as a tool to analyse gene expression in S. avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.     

幽门螺杆菌VacA重组蛋白表达、纯化及鉴定

目的研究幽门螺杆菌空泡毒素（VacA）编码基因在大肠埃希菌中的表达及纯化重组蛋白的抗原性。方法将PET32a-vacA-E.coli BE21（DE3）工程菌株常规培养,碱裂解法小量提取重组质粒DNA,琼脂糖凝胶电泳进行酶切鉴定,基因测序法进行插入基因序列分析。重组蛋白采用IPTG诱导表达,镍亲和层析原理提纯,ELISA法检测其抗原性。结果经酶切鉴定表明,插入的基因片段全长约2240bp,测序分析及与Genebank比较,可以肯定插入片段为vacA基因,ELISA法检测重组蛋白具有良好的抗原性。结论VacA重组蛋白在大肠埃希菌中成功表达,重组蛋白具有良好的抗原性。     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

重组昆虫病原细菌Cip蛋白基因工程菌发酵条件的初步研究

昆虫病原细菌产生两种胞内晶体蛋白CipA和CipB,为新型的生物农药——昆虫病原线虫提供必需的营养。在已构建原核表达载体pET-15b-cipA和pET-15b-cipB的基础上,研究了这两种重组载体在不同宿主菌中的表达情况,重组菌的生长特性,摇瓶培养时表达重组Cip蛋白工程菌的发酵条件。结果显示:以E.coli BL21(DE3)为宿主菌,在LB培养基中培养至OD600为0.8～1.0时,1 mmol/L IPTG诱导8 h,CipA和CipB的表达量可达30.9%和32.6%,重组质粒具有良好的分离稳定性和结构稳定性。     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究

[目的]右旋糖酐蔗糖酶是一种以蔗糖为底物,催化转移D-葡萄糖基生成α-葡聚糖或低聚糖的葡萄糖基转移酶.[方法]利用PCR扩增技术,将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a( )上,转化E.coli BL21(DE3),经过卡那霉素抗性筛选和酶切验证后,得到右旋糖酐蔗糖酶工程菌株BL21(DE3)/pET28-dexYG.[结果]经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制.通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察,得到最佳培养条件为:培养时间5h、IPTG浓度0.5mmol/L、25℃、OD600值1.0和pH6.0.酶活力由最初的5.39U/mL提高到35.62U/mL,其中pH值对产酶活力影响最大,在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍,并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一.[结论]酶的表达和酶活的研究结果表明,构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶,并且表现出较高的酶活力.     

Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.