乳糖作为诱导剂对重组目的蛋白表达的影响

将重组粒细胞-巨噬细胞集落刺激因子/白细胞介素3(GM-CSF/IL-3)融合蛋白表达菌BL21(DE3)(pFu)作为研究对象,对于以乳糖作为诱导剂时重组目的产物的诱导表达规律进行了深入的研究。分析比较了不同培养基中,不同生长阶段进行诱导对于产物表达的影响。对诱导所需的乳糖浓度、诱导持续时间长短等因素亦进行了研究。实验结果表明,在对诱导条件进行优化控制的前提下,利用乳糖作为诱导剂可以达到与IPTG类似的诱导效果。随后的研究中,将乳糖作为诱导剂应用于高密度发酵过程。这些研究结果为乳糖作为诱导剂最终应用于重组基因工程药物的工业化生产提供了有益的参考和借鉴。     

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.     

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。     

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。     

L-乳酸脱氢酶在大肠杆菌BL-21(DE3)中的表达

为了在大肠杆菌中构建利用葡萄糖生产L-乳酸的途径,以鼠李糖乳杆菌(Lactobacillus rhamnosus)LA - 04 -01基因组为模板,设计引物扩增L-乳酸脱氢酶基因L-ldh.将该基因连接到表达栽体pET-28a(+)上,并转化大肠杆菌Top10.通过卡那霉素抗性平板筛选,提取重组质粒pET28a-L-ldh并测序,结果正确.将pET28a-L-ldh转化大肠杆菌BL-21( DE3),通过卡那霉素抗性平板筛选,得到产乳酸的大肠杆菌基因工程菌.经IPTG诱导后,SDS-PAGE电泳,检测到目的蛋白条带,L-乳酸脱氢酶比酶活力达到9.44 U/mL.该基因工程菌通过摇瓶发酵,L-乳酸产量达到3 g/L,成功构建出一条在大肠杆菌中生产L-乳酸的新途径.     

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

绿色荧光蛋白标记的嗜水气单胞菌在温暖水体的存活及稳定性

用绿色荧光蛋白基因(GFP)标记嗜水气单胞菌4332株(Ah4332GFP),检测其在温暖水体中的存活及稳定性.在灭菌自然塘水中,Ah4332GFP存活47d之久,该菌浓度随时间而变化,呈现两个峰值;在非灭菌自然塘水中,Ah4332GFP存活18d,该菌浓度均随时间延长呈下降趋势.表明Ah4332GFP和天然细菌之间的相互作用影响其稳定性.用M9培养基检测Ah4332GFP质粒的稳定性,结果显示在传代培养40、70代后,质粒稳定率分别保持在473%和205%.可见pGFP是一种相对稳定的质粒,Ah4332GFP可用于微生物生态的研究.       

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

Production of recombinant protein in Escherichia coli cultured in extract from waste product alga,Ulva lactuca

This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six‐months‐old were able to produce two‐fold more GFP protein than traditional Lysogeny Broth media. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:784–789, 2014     

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。     

TAT-hEGF融合蛋白在E.coli BL21(DE3)中高效自我表达

为了获得TAT-hEGF融合蛋白在E.coliBL21(DE3)中高效表达,构建了原核表达载体pRSET-tat-hegf,将其转化E.coliBL21(DE3)得到重组工程菌BL21(DE3)/pRSET-tat-hegf。工程菌在无IPTG的诱导下实现了高效表达,TAT-hEGF融合蛋白的表达量占总菌体蛋白的45.6%,主要以包涵体形式存在。     

Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein

A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri-specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LT(B)) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5'-ST-LT(B)-3' DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLT(B) system was found to possess the capability of adhering to the mice gut, secreting GFP:STLT(B) product at 0.14 and 0.026 pgcell(-1) under induced and noninduced conditions, respectively. Further analysis of the GFP:STLT(B) product confirmed its ganglioside-binding ability, LT(B) antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLT(B) culture in mice elicited significant (P<0.01) serum IgG and mucosal IgA antibodies against the STLT(B) antigen. These immunized mice were subsequently challenged with ETEC and showed full protection against the fluid influx response in the gut. This is the first report of using L. reuteri as a vaccine carrier to induce complete immunologic protection against ETEC.     

人胰高血糖素样肽-1突变体融合蛋白在大肠杆菌中的自诱导表达优化

考察了在大肠杆菌中自诱导表达人胰高血糖素样肽-1突变体融合蛋白的可行性,并对自诱导培养条件及培养基成分进行优化,以提高蛋白产量。实验结果表明,最优培养基成分为蛋白胨19.17g/L,酵母膏9.59g/L,Na2HPO45.72g/L,KH2PO45.48g/L,(NH4)2SO42.66g/L,NaCl3.33g/L,甘油2%(V/V),葡萄糖0.68g/L,乳糖6.33g/L,MgSO40.24g/L。在温度33°C、接种量1%、pH7、装瓶量20mL/100mL培养条件下,用该最优培养基自诱导表达人胰高血糖素样肽-1突变体融合蛋白的产量可达348.6mg/L。     

大肠杆菌BL21(DE3)lpxM突变株的构建

目的:lpxM基因失活可以产生极低内毒素活性的脂多糖。构建大肠杆菌BL21(DE3)的lpxM突变株,并考察该突变株的生长状态和表达重组蛋白的能力。方法:构建同源臂长500bp左右的打靶载体,借助Red同源重组系统,使E.coliBL21(DE3)的lpxM基因发生插入失活,再导入编码FLP位点特异性重组酶的质粒pCP20去除抗性基因。PCR鉴定发生插入突变的菌株,应用SDS-PAGE分析突变前后的脂多糖,考查对重组蛋白表达的影响。结果:PCR鉴定结果说明lpxM基因发生了插入突变。与出发株比较,突变株的脂多糖电泳图谱发生了明显变化,但其生长状态与表达重组蛋白的能力与出发株基本一致。结论:大肠杆菌BL21(DE3)的lpxM突变株可以用于重组蛋白的表达。     

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.     

大肠杆菌cai DE的基因克隆与表达

从Ｅ．ｃｏｌｉ ＭＣ４１００菌体的染色体ＤＮＡ中利用鸟枪法克隆含编码肉碱消旋酶及相关因子的ｃａｉＤＥ基因片段,并经序列分析验证,由重组质粒ｐｌＸ３９３亚克隆得到ｐＤＳＷ２重组表达质粒,后者转入Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）菌株中是丙基硫代半乳糖苷（ＩＰＴＧ）诱导,在聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）上分子量为３０ｋＤ和２４ｋＤ附近可见明显的表达蛋白带。