Identification of <Emphasis Type="Italic">Escherichia coli</Emphasis> host cell for high plasmid stability and improved production of antihuman ovarian carcinoma × antihuman CD3 single-chain bispecific antibody

To improve the plasmid stability during the production of antihuman ovarian carcinoma × antihuman CD3 single-chain bispecific antibody (AhOC×AhCD3), the Escherichia coli BL21(DE3) host cell was optimized serially. Firstly, an isogenic recombination-deficient (recA ⁻) derivative of BL21(DE3), namely BLR(DE3), was used as host instead of BL21(DE3). Although the segregational plasmid stability was greatly improved, AhOC×AhCD3 yield was not improved due to the severe growth inhibition of plasmid-bearing BLR(DE3) cells and the competitive plasmid instability after induction. Secondly, a mutant BLR(DE3), namely BLRM(DE3), was screened by using LB agar plates plus ampicillin and isopropyl-β-d-thiogalactopyranoside. Using this new host, growth inhibition of recombinant cells after induction was eliminated, and plasmids could be stably maintained even after long-time induction in a nonselective medium. At last, about 1.2 g/l AhOC×AhCD3, which was about thrice as much as those of recombinant BL21(DE3) and BLR(DE3) strains, was yielded.     

Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 degrees C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.     

无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测

目的:利用基因工程的方法原核表达无标签的重组人硫氧还蛋白(rhTrx)并对其进行大规模表达、纯化和鉴定.方法:从人胚胎肾HEK293细胞中提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒,重组质粒转化大肠杆菌BL21( DE3)感受态细胞,IPTG诱导表达,经两步离子交换层析纯化重组蛋白,采用SDS-PAGE、Western blotting、HPLC、MALDI-TOF-MS及经典的胰岛素二硫键还原法对重组蛋白进行鉴定.结果:构建成功了rhTrx基因表达载体;实现了rhTrx在原核细胞中的可溶性表达;纯化出的蛋白经SDS-PAGE和Western blotting分析证实为rhTrx;HPLC和MALDI-TOF-MS分析表明,纯化出的目的蛋白纯度大于95％;胰岛素二硫键还原法证实纯化出的rhTrx具有生物学活性.结论:成功构建了rhTrx的原核表达体系,建立了rhTrx的纯化和鉴定方法,为其进一步的理论研究和生产开发提供了有效基础数据.     

Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membrane structure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high‐ and low‐strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5‐1A and lac‐1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3‐lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale‐up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3‐lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.     

重组尿酸氧化酶发酵工艺优化

目的:优化并获得重组尿酸氧化酶(rUOX)基因工程大肠杆菌BL21(DE3)/pET-32a-uox高密度发酵的工艺参数。方法:在三角摇瓶中进行培养条件的优化实验,分别考察了pH值、接种量、无机盐、碳源、诱导强度等对工程菌生长和重组蛋白表达的影响,得到了优化的发酵条件;在此基础上放大至NBS BIOFLO 110 14 L发酵罐,通过对诱导时机的优化,利用分批补料发酵的方式,使rUOX在高密度培养的条件下得到高表达。结果:在优化的发酵条件下,菌体密度(D600nm)最终达到50以上,相当于20 g/L干重;可溶性rUOX占菌体总蛋白量的45%,其含量达到3.45 g/L。结论:为规模化制备重组黄曲霉尿酸氧化酶奠定了基础。     

Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli

In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l⁻¹.     

还原酶缺陷型大肠杆菌对重组蛋白溶解性的影响

探讨大肠杆菌细胞质氧化还原环境对重组蛋白溶解性的影响。选择含有1对二硫键的牛碱性成纤维细胞生长因子（BbFGF）作为简单蛋白的模式分子,选择含有2对二硫键的人抗HBsAg单链抗体（HBscFv）作为复杂蛋白的模式分子,分别构建表达质粒并转化普通宿主菌和还原酶缺陷型宿主菌E. coli Origami(DE3),比较表达产物的溶解性和纯化产物的活性。结果发现,BbFGF在普通宿主菌中大部分形成包涵体,在Origami(DE3)中为可溶性表达,但表达量降低。两种工程菌的表达产物经离子交换和肝素亲和层析两步纯化后,MTT法测定活性,发现来自还原酶缺陷型宿主菌的BbFGF活性高于普通宿主菌表达产物,二者的ED₅₀分别是1.6 ng/mL和2.2 ng/mL;HBscFv在两种宿主菌中均形成包涵体,包涵体以6 mol/L盐酸胍缓冲液溶解后,镍离子螯合亲和层析纯化并透析复性,间接ELISA测定抗原结合活性,发现二者活性无明显差异,但在Origami(DE3)菌体破碎后的的上清中可检测到HBscFv活性,纯化后产量为1～2 mg/L,而在普通宿主菌破碎后的上清中检测不到HBscFv活性。上述结果说明,改变宿主菌细胞质氧化还原环境对于含有1～2对二硫键的重组蛋白的可溶性表达具有明显促进作用。     

Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.     

Angioarrestin(hARPl)C端FD区的克隆、表达及其免疫活性鉴定

 Angioarrestin是一种具有潜在应用价值的肿瘤血管形成抑制因子.利用DNA重组法构建了angioarrestin C端 hFD cDNA 和麦芽糖结合蛋白(MBP)重组原核表达质粒 pMAL-C2-hFD.将重组质粒转入大肠杆菌E.coli BL21（DE3）,经0.3 mmol/LIPTG 在37℃条件下诱导表达4h,SDS-PAGE 检测,融合蛋白表达量约占细菌总蛋白的20%.Western印迹证实,目的蛋白N端带有MBP标签.取表达上清纯化、透析、浓缩并冻干,以此为抗原免疫Balb/c小鼠制备多克隆抗体.此多抗可以与pET 22b（+）表达系统获得的 hFD重组蛋白发生良好的抗原抗体反应,ELISA检测多抗效价达1∶10240.实验证明：通过基因重组可获得angioarrestin C端hFD在大肠杆菌中的高效表达蛋白,且该蛋白具有较高的免疫活性.以此为抗原制备的抗angioarrestin多克隆抗体为深入研究angioarrestin提供了材料.     

Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (lambdaDE3) and Escherichia coli JM109

Two Escherichia coli strains, widely used for the production of various recombinant proteins, were compared for their pre-induction growth and acetate accumulation patterns. The strains studied were E. coli BL21 (lambdaDE3), transformed with a plasmid encoding Pseudomonas exotoxin A, and an E. coli K12 derived strain, JM109, carrying a plasmid encoding maltose-binding protein fused with HIV protease. Cultures were grown in controlled bench-top fermentors to the optimal pre-induction density in both high glucose batch and low glucose fed batch strategies. The results showed the superiority of E. coli BL21 (lambdaDE3) as a host for a recombinant protein expression system. For example, JM109 responds differently to high glucose concentration and to low glucose concentration. Its acetate concentration was as high as 10 g/L in a batch mode and 5 g/L in a fed batch mode. In comparison, strain BL21 (lambdaDE3) reached 2 g/L acetate when grown in batch mode and not more than 1 g/L acetate when grown in a fed batch mode. E. coli BL21 (lambdaDE3), most likely, possesses an acetate self-control mechanism which makes it possible to grow to the desired pre-induction density in a high glucose medium using simple batch propagation techniques. Such a technique is cost effective, reproducible, and easy to scale up. (c) 1996 John Wiley & Sons, Inc.     

颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达

为高效表达颗粒裂解肽G13结构域并避免G13对宿主菌的毒性, 将人工合成的编码G13的基因片段, PCR扩增后克隆于原核表达载体pThioHisA中, 构建了重组表达载体pThioHisA-G13, 将其转化于大肠杆菌BL21(DE3)中, 经IPTG诱导表达融合蛋白Trx-G13, 表达产物以包涵体的形式存在, 其表达量约占细菌总蛋白的58%。包涵体蛋白经 8 mol/L尿素溶解后, 再经CNBr切割, 阳离子交换层析, 得到纯化的重组G13结构域。琼脂糖扩散法检测表明重组G13结构域多肽具有抗菌活性。     

Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli

It is difficult to express recombinant Candida rugosa lipases (CRLs) in heterologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG for leucine. In this study, recombinant LIP4 in which all 19 CUG codons had been converted to a universal serine codon was overexpressed in Escherichia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion bodies and showed a low catalytic activity. To increase the amount of soluble form and activity of recombinant LIP4, the DNA was fused to the gene for thioredoxin (TrxFus-LIP4) and then expressed in E. coli strain AD494(DE3). This strategy promotes the formation of disulfide bonds in the cytosol and yields enzymatically active forms of LIP4. The purified recombinant TrxFus-LIP4 and LIP4 expressed in AD494(DE3) had the same catalytic profiles. In addition, recombinant LIP4 had higher esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein, and olive oil. This system for the expression of fungal lipase in E. coli strain AD494(DE3) is reliable and may produce enzymatically active forms of recombinant lipase without an in vitro refolding procedure.     

重组大肠杆菌全细胞催化合成莱鲍迪苷D

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。     

Expression of human cardiac-specific homeobox protein in Escherichia coli

Human cardiac-specific homeobox protein cDNA (hCsx) was cloned into expression plasmid pET32a and fused with Escherichia coli thioredoxin (Trx). The Trx-Csx fusion protein was under the control of bacteriophage T7 promoter. When expressed in E. coli BL21(DE3), about half of the recombinant Trx-Csx products existed in the form of insoluble inclusion bodies. When coexpressed with human protein disulfide isomerase, more than 90% of Trx-Csx products accumulated in the soluble form in the cell lysate. The recombinant Csx fusion protein was purified by one-step metal-chelating affinity chromatography.     

High-level expression of a soluble and functional fibronectin type II domain from MMP-2 in the Escherichia coli cytoplasm for solution NMR studies

We report a method for the expression in Escherichia coli of the isolated second type II fibronectin domain from MMP-2 (FNII-2). FNII-2 was expressed as a His(6)thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation. When cultured at 37 degrees C, the expressed protein is located exclusively in the soluble fraction of the E. coli lysate. The fusion protein from the soluble fraction was purified and the His(6)thioredoxin-tag was cleaved by thrombin, resulting in a yield of approximately 40 mg/L. The recombinant FNII-2 was demonstrated to be functional by its ability to bind to gelatin-Sepharose, correct folding of the purified protein was confirmed by NMR spectroscopy. This approach may generally be applicable to all FNII domains and is a significant simplification relative to existing techniques involving refolding from inclusion bodies or expression in the eukaryotic host, Pichia pastoris.     

表达重组葡激酶-水蛭素融合蛋白的大肠杆菌工程菌的高密度培养

采用溶氧反馈的分批培养流加补料的方法高密度培养重组大肠杆菌BL21(DE3)生产重组葡激酶-水蛭素融合蛋白。通过摇瓶培养对菌种和培养条件的初步筛选,采用溶氧反馈的流加补料策略,进行了5L发酵罐的合成培养基和复合培养基的发酵工艺的研究。通过对培养条件的不断优化,重组葡激酶-水蛭素融合蛋白在大肠杆菌BL21(DE3)里得到了高效表达,菌体密度最终达到115g/L(WCW)以上,可溶性重组融合蛋白占菌体总蛋白的30%以上,含量约为1.1~1.2g/L。5L发酵罐的发酵工艺参数在40L发酵罐中进行了放大培养,结果表明该工艺能有效的放大,可适用于工业生产。     

液化沙雷氏菌磷脂酶A1的克隆表达及乳糖自诱导发酵

为了利用大肠杆菌高效生产重组磷脂酶,克隆了液化沙雷氏菌磷脂酶A1的编码基因pla,分别使用pET-28a(+)和pET-20b(+)载体,实现了磷脂酶A1在大肠杆菌BL21(DE3)中的功能表达.重组菌利用载体pET-28a(+)在原始信号肽的介导下胞外PLA1酶活达40.8 U/mL,占总酶活的91％.重组菌转接至优化后的发酵诱导培养基:蛋白胨10 g/L,酵母粉5g/L,葡萄糖0.8 g/L,乳糖5 g/L,25 mmol/L Na2HPO4,25 mmol/L KH2PO4和1 mmol/L MgSO4;菌体生长6h后,添加7.5 g/L的甘氨酸,37℃恒温发酵24 h,重组菌胞外PLA1酶活达到128.7 U/mL.     

钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究

目的:对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF(SYPO-1)进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法:通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果:来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg~(2+)、Li~+、Mn~(2+)促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/(L·h)。结论:筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum(SYPO-1)中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。     

以重组P-gp为抗原建立检测MDR 1抗体间接ELISA方法的研究

目的:构建MDR 1基因原核表达质粒,表达P-gp重组蛋白,建立检测MDR1抗体的间接ELISA方法。方法:利用重组PCR技术扩增MDR 1基因的1kb片段,克隆至pET-28b（＋）中,构建原核表达质粒pETP-gp,转染感受态菌BL21（DE3）和BL21（DE3）plyss;以E.coli高效表达的P-gp基因主要抗原编码区重组蛋白为抗原,以HRP标记的兔抗人IgG为二抗,建立间接ELISA检测方法。结果:正确构建了pETP-gp原核表达质粒,并可在E.coli中高效表达,表达蛋白可用作检测MDR 1抗体ELISA抗原。结论:成功表达出重组蛋白P-gp,建立了检测MDR 1抗体的间接ELISA方法。     

High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli

The entire encoding region for Aspergillus flavus uricase was cloned into pET-32a and expressed in Escherichia coli BL21 (DE3). The uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form. A scalable process aimed to produce and purify multi-gram quantities of highly pure, recombinant urate oxidase (rUox) from E. coli was developed. The rUox protein was produced in a 30 L fermentor containing 25 L of 2x YT medium and purified to >99% purity using hydrophobic interaction, anion-exchange, and gel filtered chromatography. The final yield of purified rUox from fermentation resulted in approximately 27 g of highly pure, biologically active rUox per kg of cell paste (approximately 238 mg/8.8 g cell paste/L). The results presented here exhibit the ability to generate multi-gram quantities of rUox from E. coli that may be used for the development of pharmaceutics of reducing the hyperuricemia.