Effects of pretreated beet molasses on benzaldehyde lyase production by recombinant Escherichia coli BL21(DE3)pLySs

Aims: To investigate the effects of pretreated‐beet molasses on Escherichia coli fermentation using benzaldehyde lyase (BAL) production by recombinant E. coli BL21(DE3)pLySs process as the model system. Methods and Results: The effect of the initial pretreated (hydrolysed) beet molasses concentration was investigated at 16, 24, 30 and 56 g l^?1 at a dissolved oxygen condition of 40% air saturation cascade to airflow, at N = 625 min^?1 and pH_C = 7·2 controlled‐pH operation conditions. The highest cell concentration and BAL activity were obtained as C_X = 5·3 g l^?1 and A = 1617 U cm^?3, respectively, in the medium containing 30 g l^?1 pretreated beet molasses consisting of 7·5 g l^?1 glucose and 7·5 g l^?1 fructose. Production with and without IPTG (isopropyl‐β‐d ‐thiogalactopyranoside) induction using the medium containing 30 g l^?1 of pretreated beet molasses yielded the same amount of BAL production, where the overall cell yield on the substrate was 0·37 g g^?1, and the highest oxygen transfer coefficient was K_La = 0·048 s^?1. Conclusions: Pretreated beet molasses was used in the fermentation with E. coli for the first time and it yielded higher cell and BAL production compared with the glucose‐based medium. Significance and Impact of the Study: Pretreated beet molasses was found to be a good carbon source for E. coli fermentation. Furthermore, IPTG addition was not required to induce recombinant protein production as galactose, one of the monomers of trisaccharide raffinose present in the beet molasses (1·2%), induced the lac promoter.     

利用指数流加补料高密度发酵产β-呋喃果糖苷酶重组大肠杆菌

【目的】对重组大肠杆菌BL21(DE3)/pET22b-β-ffase进行高密度发酵产β-呋喃果糖苷酶工艺研究。【方法】比较溶氧反馈补料和指数流加补料对重组菌发酵产酶的影响,对不同比生长速率和诱导时机进行优化。【结果】确定了双阶段指数流加过程中重组菌生长的比生长速率,分别控制诱导前期比生长速率为0.20 h~(-1),诱导后期比生长速率为0.13 h~(-1),诱导时机为指数中期。获得细胞干重约为51 g/L,最高酶活达到1.79×10~5 U/L,单位菌体产酶量为3 510 U/g,单位产酶速率达到3.58×10~4 U/(L·h),生物量、单位菌体产酶量和产酶速率分别是指数流加未优化前的1.8、1.7和3.0倍。【结论】双阶段指数流加补料工艺能有效提高β-呋喃果糖苷酶的产酶量,为β-呋喃果糖苷酶的进一步工业化奠定基础。     

Use of a derivative of Escherichia coli BL21(DE3) for efficient production of three different recombinant proteins

Plasmid instability and growth inhibition of plasmid-bearing cells after induction were encountered when E. coli BL21(DE3) was used as host for the production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody (AhOC x AhCD3), human soluble B lymphocyte stimulator fused with thioredoxin (Trx-hsBLyS) and human parathyroid hormone fused with thioredoxin (Trx-hPTH). A derivative of BL21(DE3), namely, BLRM(DE3), isolated and showing superiority in AhOC x AhCD3 production in our previous work, was further used here for more efficient production of these three different recombinant proteins. By using BLRM(DE3) as host, the simplified one-stage fermentation process was developed, which was more labor-saving and yielded AhOC x AhCD3, comparable to that of the traditional two-stage fermentation process. Also, the plasmid stabilities and production yields of Trx-hsBLyS and Trx-hPTH were dramatically improved by the application of BLRM(DE3) instead of BL21(DE3). A high Trx-hsBLyS yield (about 3.5 g/L) was obtained, which was more than twice as much as that of the recombinant BL21(DE3) strain. The Trx-hPTH yield was improved from about 700 mg/L to 1 g/L. These results further showed the superiority of BLRM(DE3) to BL21(DE3) and suggested its effectiveness for other BL21(DE3)/pET heterologous protein expression systems, which encounter similar problems.     

乳糖代替IPTG诱导己糖激酶在大肠杆菌中的表达

构建了己糖激酶GLK高效表达的工程菌株BL21(DE3)/pET-glk,考察了乳糖代替IPTC诱导己糖激酶GLK在大肠杆菌BL21(DE3)中表达的可行性.实验结果表明,工程菌对数生长中期(OD<,600>约为1.0)添加终浓度为10 g/L的乳糖于25℃的条件下诱导6 h能获得最大量的目的蛋白和菌体量,目的蛋白表达...     

大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21 (DE3)染色体上的基因waaF或msbB敲除,构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G 酰化酶(penicillin G acylase,PGA)基因的重组质粒pET-ffase、pET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株ΔmsbB为宿主时,胞外分泌量达到19.7%,而以敲除菌株ΔwaaF为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株ΔwaaF为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株ΔmsbB和ΔwaaF,ΔmsbB能明显增强β-FFase的胞外分泌,而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。     

大肠杆菌BL21(DE3)磷酸转乙酰基酶缺陷变株的发酵研究

研究了E.coliBL21(DE3)及其磷酸转乙酰基酶(PTA)缺陷变株FR55发酵过程中菌体生长和有机酸产生情况,并以肿瘤坏死因子(TNF)为外源蛋白表达的模型考察了pta基因缺陷对外源蛋白表达的影响。在摇瓶培养条件下,pta变株TNF的表达水平比亲株提高了23%。在5L发酵罐中进行了补料分批培养试验,在不限制比生长速率的条件下pta变株能够以较长时间和较高比生长速率保持对数生长,最终达到32.5g(DCW)/L的菌密度,TNF的总表达量达2.8g/L;而在相同条件下,以BL21(DE3)为受体菌的对照组最高菌密度为19.5g(DCW)/L,TNF总表达量只有0.84g/L。表明pta变株对于提高工程菌外源蛋白的表达和实现高密度培养具有一定应用价值。分析了补料分批培养过程中发酵液有机酸组成和含量的动态变化情况,发现pta变株乙酸累积水平明显降低(为亲株乙酸累积水平的42%)的同时,其他几种有机酸(丙酮酸、乳酸、琥珀酸)的累积有显著增加的趋势,使发酵液中总有机酸浓度增加了123%,其中乳酸的累积是影响菌体进一步生长的主要因素。     

Proteolysis and synthetic strategy of human G-CSF in Escherichia coli BL21(DE3)

We report for the first time that the C-terminal region of hG-CSF suffers from proteolytic degradation when human granulocyte colony-stimulating factor (hG-CSF) is directly expressed in Escherichia coli BL21(DE3). It is believed that the rapid proteolysis occurs at the C-terminus of hG-CSF that is very easily exposed to E. coli protease(s) during a short period following protein synthesis and prior to completion of the formation of the inclusion body. The recombinant hG-CSF that is expressed with an N-terminal fusion partner is effectively protected from the proteolysis. It seems that since the N-terminus of hG-CSF is located very close to the C-terminus, the presence of the N-terminal fusion partner masks the C-terminal region of hG-CSF and protects it from proteolytic degradation by E. coli protease(s). Furthermore, the solubility of hG-CSF markedly increased in E. coli cytoplasm when a stress-responsive and aggregation-resistant protein, i.e. aspartate carbamoyl-transferase catalytic chain (PyrB) was used as a novel N-terminal fusion partner proteins.     

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.     

STATISTICAL OPTIMIZATION OF GREEN FLUORESCENT PROTEIN PRODUCTION FROM Escherichia coli BL21(DE3)

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box–Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD_600nm) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.     

The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3)

Two mutant strains of Escherichia coli BL21(DE3), called C41(DE3) and C43(DE3) and originally described by Miroux and Walker, are frequently used to overcome the toxicity associated with overexpressing recombinant proteins using the bacteriophage T7 RNA polymerase expression system. Even when the toxicity of the plasmids is so high that it prevents transformation in the strain BL21(DE3), the toxic proteins can often be expressed successfully in C41(DE3) and/or C43(DE3). In this work, using a range of plasmids coding for several types of proteins, we investigated in BL21(DE3), C41(DE3), and C43(DE3) their ability to undergo transformation and to express. While transformation was always possible in C41(DE3) and C43(DE3), we could not obtain transformants in BL21(DE3) for 62% of the expression vectors tested. Moreover, after induction, the expression of heterologous proteins in both mutant strains is generally better than in BL21(DE3). In this study, we also enhanced the stability of plasmids in culture during the expression of proteins by adding the par locus from the plasmid pSC101 to the vector backbone. The stability of a subset of the plasmids (measured 3 h after induction) was determined in C41(DE3) and C43(DE3) and varies from 62 to 92% for C43(DE3) and from 10 to 90% for C41(DE3). This study demonstrates the usefulness of these strains C41(DE3) and C43(DE3) in solving the problem of plasmid instability during the expression of toxic recombinant proteins.     

Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

鼠疫菌V抗原基因在大肠杆菌中的克隆及表达

从鼠疫杆菌野毒株总DNA中利用PCR方法扩增出V抗原编码基因,将此基因克隆到大肠杆菌的表达质粒pET42b(+)中,经IPTG诱导后,在大肠杆菌BL21(DE3)细胞中成功地表达出鼠疫菌V抗原蛋白。目的蛋白表达量占菌体总蛋白的32.8%。     

rimJ基因缺失不影响大肠杆菌BL21(DE3)生长的温度敏感性

目的:研究rimJ基因对大肠杆菌BL21(DE3)菌株生长的温度敏感性的影响。方法:利用SceⅠ-Red同源重组技术将大肠杆菌BL21(DE3)基因组中的rimJ基因缺失,得到rimJ缺失突变菌;比较缺失突变株和原始菌株在不同温度(25℃、37℃、42℃)下的最大比生长速率,利用统计学方法分析rimJ基因的缺失对BL21(DE3)菌株生长的温度敏感性是否有影响。结果:rimJ基因被成功敲除;统计分析结果表明缺失突变株和原始菌株的最大比生长速率没有明显区别。结论:rimJ基因缺失对大肠杆菌BL21(DE3)生长的温度敏感性无影响。     

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.     

Effect of iclR and arcA deletions on physiology and metabolic fluxes in Escherichia coli BL21 (DE3)

Deletion of both iclR and arcA in E. coli profoundly alters the central metabolic fluxes and decreases acetate excretion by 70%. In this study we investigate the metabolic consequences of both deletions in E. coli BL21 (DE3). No significant differences in biomass yields, acetate yields, CO₂ yields and metabolic fluxes could be observed between the wild type strain E. coli BL21 (DE3) and the double-knockout strain E. coli BL21 (DE3) ΔarcAΔiclR. This proves that arcA and iclR are poorly active in the BL21 wild type strain. Noteworthy, both strains co-assimilate glucose and acetate at high glucose concentrations (10–15 g l⁻¹), while this was never observed in K12 strains. This implies that catabolite repression is less intense in BL21 strains compared to in E. coli K12.     

含谷胱甘肽硫转移酶基因工程菌表达条件研究

将重组谷胱甘肽硫转移酶 (GST)大肠杆菌表达株BL2 1(DE3 )PGEX 作为研究对象 ,进行了生长及表达条件的优化研究 ,分析比较了不同培养条件对菌体生长的影响以及诱导剂的添加量对产物表达量的影响。