重组大肠杆菌分批-补料高密度发酵生产类人胶原蛋白的动力学

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.     

重组类人胶原蛋白的分离纯化

目的 建立重组类人胶原蛋白分离及纯化工艺。方法 将工程菌株E.coli BL21 3.1在L1523型12.8L自控发酵罐中培养14h,发酵菌体经超声破碎、硫酸铵沉淀后,依次经DEAE-52和Sephadex G-100柱层析分离纯化。结果 发酵菌体干重达到71g/L,表达量为菌体蛋白量的29.4％,最终产物纯度达98％,回收率为80.5％,纯化倍数为3.4。结论 纯化的类人胶原蛋白达电泳纯,相对分子质量为90 000,N端序列为NH_2-H-D-P-V-V-L-Q-R-R-D-W-E-N-P-G,均与其因序列设计一致。     

基因重组大肠杆菌生产类人胶原蛋白补氮策略优化研究

优化重组大肠杆菌高密度发酵的调控工艺.利用重组大肠杆菌高密度发酵生产类人胶原蛋白,考察了连续流加和分阶段式两种不同补氮策略对细胞生长和类人胶原蛋白产量的影响.不同补氮方式显著影响类人胶原蛋白的产量.分阶段式补氮方式优于连续流加补氮方式,有利于细胞生长和类人胶原蛋白的表达,最终细胞浓度为76.01 g·L-1(DCW),类人胶原蛋白浓度为16.75 g·L-1,蛋白表达量为22.1%.     

大肠杆菌E.coli HB101(pBR322) 高密度培养过程质粒的稳定性

通过正交实验考察了大肠杆菌E.coli HB101(pBR322)高密度培养过程中,不同培养条件对质粒的稳定性和β-内酰胺酶活力的影响. 结果表明,当温度在33~39℃、pH为6.4~7.2、DO为 40%~80%、 补料速率在5.4~10.8 g/h范围内变化,以及细胞密度达到27.3 g/L、比生长速率达到0.73 h-1时,质粒没有发生丢失现象,但在培养过程中β-内酰胺酶的活力降低.     

重组类人胶原蛋白研究应用进展

总结了目前国内主流重组类人胶原蛋白原料生产和新产品开发团队的最新研究成果,介绍了重组类人胶原蛋白的研究热点,对未来发展进行展望.     

重组人源型胶原蛋白基因工程菌补料分批发酵过程动力学研究

研究了重组人源型胶原蛋白基因工程菌在分批发酵阶段和补料分批发酵阶段的生长特性,得到在分批发酵过程菌体生长动力学方程dρBρB和基质消耗动力学方程-dρSρB+0.05ρB;在dt=0.67ρS0.95+ρSdt=0.45ρS0.95+ρS补料分批发酵阶段,利用近似指数流加培养方式进行补料,得到菌体干重为71.02g·L-1;归纳的模型与实际发酵过程的实验数据吻合良好。     

种子扩大培养过程对类人胶原蛋白规模化生产的影响

为了建立最优的类人胶原蛋白(HLC)的种子扩大培养过程,考察了三级种子培养过程中不同移种阶段和不同种子培养基对发酵过程的影响。结果表明,在对数期后期移种,HLC产率最高;当种子培养基中葡萄糖浓度为20 g·L-1时,其后发酵过程所需的培养时间较短,HLC表达量较高,HLC平均产率最高,达到0.518 g·L-1·h-1...     

分泌型重组人生长激素工程菌的高密度发酵

目的　研究葡萄糖剂补加策略 ,以获得大肠杆菌的高密度发酵和人生长激素 (HGH)的高表达。方法　采用批式发酵和补料批式发酵方式 ,葡萄糖的补加采用逐渐增加的方式 ,包括pH、DO和浓度控制。结果　获得HGH的表达量为 30 0mg/L ,光密度达到 12 4(A60 0 )。结论　在大肠杆菌中表达异源蛋白时 ,葡萄糖的补加方式是关键因素 ,补料批式发酵要优于批式发酵     

重组人碱性成纤维细胞生长因子工程菌高密度发酵及表达产物的纯化

目的 研究重组人碱性成纤维细胞生长因子工程菌高密度发酵和表达产物的纯化工艺。方法采用最适基质浓度、pH、溶氧量、培养温度等发酵表达条件及产物提纯方法。结果 菌体密度A6000达40.8,表达量达160mg/L。提纯的bFGF回收率为40％,生物学活性（ED50）为 0.21ng/mL,比活性达4.76×106U/mg,其纯度、分子量、残余DNA等各项检测结果均符合“重组 DNA制品质量控制要点”。结论 为hbFGF工业化生产和临床应用提供了依据。     

E.coli M15 (pQTPL)高效发酵生产酪氨酸酚裂解酶的控制策略

在摇瓶和4 L发酵罐上研究了营养和环境条件对重组菌E. coli M15 (pQTPL)分批发酵生产酪氨酸酚裂解酶(TPL)的影响. 在培养基中添加20 g/L葡萄糖和1.0 g/L玉米浆使TPL酶活提高到63.1 U/g(干重). 在此基础上,维持发酵液中溶氧水平为30%,可使菌体浓度在8 h达到4.78 g/L,酶活为54.6 U/g,比对照组(不控制溶氧)分别提高了21%和31.6%. 采用溶氧反馈调节-限制性补料策略,可使菌体浓度提高到31.5 g/L. 采用两阶段温度和pH控制策略,在发酵前8 h控制pH 7.0、温度37℃,8 h 至发酵结束之间控制pH为8.0、温度为30℃,可使重组菌的TPL酶活达到154.4 U/g,并使TPL在细胞中过量表达,实现了高菌体浓度和高TPL酶活的统一.     

大孔离子交换树脂在重组大肠杆菌生产hEGF中的原位分离作用

引　言乙酸是重组大肠杆菌培养过程中的主要代谢副产物[1,2 ] ,乙酸的产生和积累对重组菌的生长和外源蛋白表达都有严重的抑制作用[3,4 ] .许多研究者对此开展了多方面的研究 ,如通过代谢工程手段构建乙酸酶缺陷菌株作为宿主菌来减少乙酸的产生[5] 、采用发酵 /分离耦合技术除去或降低乙酸量[6～ 8] 、透析培养技术[9,10 ] 、控制菌体的比生长速率及限制基质葡萄糖浓度等方法 .　　　本实验室开展了重组大肠杆菌分泌性高效表达人表皮生长因子 (humanepidermalgrowthfactor ,hEGF)的研究 ,发现在重组菌培养过程中乙酸的积累对重组菌生长和hEGF表达有严重抑制作用 .本文开展了高效吸附乙酸的离子交换树脂筛选 ,并在摇瓶和发酵罐规模两个水平上研究离子交换树脂在重组大肠杆菌发酵生产hEGF过程中对乙酸的原位分离作用 .1　材料与方法1 1　菌株与质粒E coliJM10 1/pSP10 3,本研究所保存的高效分泌型hEGF的重组大肠杆菌 .1 2　培养基及培养方法种子培养基和发酵培养基 ,种子培养、摇瓶培养及 2 5LB Braun发酵罐培养的条件参照文献[11],其中发酵培养基中...     

A PURIFICATION PROCESS BASED ON SELF-ASSEMBLY FOR RECOMBINANT HUMAN-LIKE COLLAGEN

Human-like collagen (HLC) is industrially produced by using recombinant Escherichia coli and a high-cell-density fermentation technique. Similar to the collagen extracted from animal tissue, industrially produced HLC is difficult to purify because of its self-assembling property, whereby it forms aggregates of different sizes. In this study, we report the development of a purification process based on this property of HLC. The interactions of HLC fibrils were analyzed under different conditions. HLC was immobilized on Sepharose Cl-6B (HLC-Sepharose) by covalent cross-linking. This immobilization triggered lateral molecular packing of free HLC in a specific manner in a solution. We performed a purification process based on hydrogen bonding and conducted microscopic and electrophoretic analyses to assess the results of the process. We eventually obtained an electrophoretically pure product. The results show that hydrogen bonding is the main driving force that promotes the aggregation of HLC monomers, while hydrophobic bonding facilitates the stabilization of HLC aggregates. This “self-purification” method is a feasible and simple approach.     

重组大肠杆菌分批-补料高密度发酵生产类人胶原蛋白的动力学

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and growth rates were set at (0.15, 0.2, 0.25h-1) by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase spectively. The model predictions are in good agreement with the experimental data.     

诱导条件对重组大肠杆菌发酵中人白细胞介素6表达量的影响

对重组人白细胞介素6 (rhIL-6)大肠杆菌摇瓶发酵工艺作了研究.探讨了诱导策略对菌体生长和rhIL-6表达量的影响.结果表明,含有温敏扩增型质粒pKpL3a的重组hIL-6大肠杆菌,在2×YT培养基中发酵,接种量为0.2%,于30℃,180 r/min摇瓶中培养,当菌密度OD 600约为1.5时,转至42℃或40℃诱导,维持时间至少为1 h,然后转至37℃培养3 h,最终rhIL-6表达量可达32%.于40℃或42℃下诱导时间低于1 h,则使最终rhIL-6表达量降低 .如果采用42℃或40℃恒温诱导,rhIL-6表达量仅为20%.     

大肠杆菌高密度培养研究进展

对大肠杆菌高密度培养的影响因素、培养方法等方面的最新进展进行了综述,并对基因工程技术在大肠杆菌高密度培养中的最新应用进行了介绍。     

DESIGN OF RECOMBINANT PROTEIN PRODUCTION PROCESSES

A model-aided process design and supervision technique is proposed. Its efficiency is demonstrated by a nontrivial example, the development of a production process for the light chain of the antibody MAK33. This protein was expressed by the genetically modified E. coli B pUBS520 p12023 bacteria under the control of the tac promoter where lactose was used for induction. It is shown in this example that data from a few experiments are sufficient to develop a satisfactory production process. The development of process models, which are prerequisite for a systematic optimization of protein production processes, as well as their use for the determination of quasi-optimal feed-forward control profiles, is discussed. Model-supported closed-loop control is shown to lead to another improvement. Further performance enhancements can be obtained by changing the operational mode from the usually employed fed-batch procedure to a repeated fed-batch mode.     

DESIGN OF RECOMBINANT PROTEIN PRODUCTION PROCESSES

A model-aided process design and supervision technique is proposed. Its efficiency is demonstrated by a nontrivial example, the development of a production process for the light chain of the antibody MAK33. This protein was expressed by the genetically modified E. coli B pUBS520 p12023 bacteria under the control of the tac promoter where lactose was used for induction. It is shown in this example that data from a few experiments are sufficient to develop a satisfactory production process. The development of process models, which are prerequisite for a systematic optimization of protein production processes, as well as their use for the determination of quasi-optimal feed-forward control profiles, is discussed. Model-supported closed-loop control is shown to lead to another improvement. Further performance enhancements can be obtained by changing the operational mode from the usually employed fed-batch procedure to a repeated fed-batch mode.     

含青霉素酰化酶的重组大肠杆菌的发酵培养

研究了含青霉素酰化酶的重组菌 Ｅ．ｃｏｌｉ Ａ５６ （ｐ Ｐ Ａ２２）发酵过程中环境因素对酶活的影响,考察了发酵液性质变化与酶活表达之间的关系,获得了酶活较高时的优化环境条件。结果表明,青霉素酰化酶在重组菌生长的平衡期后开始大量表达,苯乙酸作为限制性底物对青霉素酰化酶的合成起诱导作用