鲤鱼生长激素基因工程菌的发酵研究

对表达鲤鱼生长激素的大肠杆菌工程菌的发酵条件进行了详细的研究,探讨了发酵条件对工程菌外源蛋白表达量的影响,优化了影响工程菌发酵的各种条件,形成了一套工程菌小量发酵表达外源蛋白的工艺,为工程菌的大量生产奠定了基础     

基因工程菌的发酵研究

本文对大肠杆菌表达的rhGM - CSF 工程菌的发酵条件进行了详细的研究, 探讨了发酵条件对工程菌表达外源蛋白量的影响, 优化了影响发酵的各种条件, 形成了一套工程菌发酵表达外源蛋白的工艺, 并从工业化角度对工程菌的高密度高表达间的关系进行了探讨。     

基因工程菌的发酵研究

本文对大肠杆菌表达的ｒｈＧＭ－ＣＳＦ工程菌的发酵条件进行了详细的研究,探讨了发酵条件对工程菌表达外源蛋白量的影响,优化了影响发酵的各种条件,形成了一套工程菌发酵表达外源蛋白的工艺,并从工业化角度对工程菌的高密度高表达间的关系进行了探讨。     

重组人血小板生成素融合蛋白工程菌发酵工艺的研究

对表达重组人血小板生成素融合蛋白（rhTPO/GST)工程菌的发酵条件进行了较详细的研究,优化了影响工程菌发酵的条件,探讨了发酵条件对工程菌表达外源蛋白量的影响。结果发现采用复合培养基分阶段流加有机营养物,用异丙基硫代-B-D-半乳糖苷（IPTG）进行诱导,使rhTPO/GST融合蛋白的湿菌重达25g/L以上,表达水平约占菌体总蛋白的30%左右。在此基础上,建立了中试发酵生产工艺流程。     

影响重组工程菌发酵产率的因素

随着分子生物学对表达外源基因的重组工程菌的研究的不断深入以及重组ＤＮＡ技术的发展,为我们更科学而不是经验地研究重组工程菌的发酵规律提供了理论基础。但有关微生物生理与质粒拷贝数、质粒稳定性和外源基因表达之间相互关系的研究还较少。本文重点综述了质粒拷贝数、质粒稳定性及外源基因表达水平等因素及环境条件对发酵过程中目标产物产率的影响,同时探讨了在工程菌发酵中以上因素的控制方法。     

鱼重组生长因子酵母的发酵研究

对含鲑鱼生长激素基因的甲醇酵母进行了发酵研究,比较了摇瓶发酵和自控罐发酵两种条件下酵母工程菌的生长和外源蛋白的表达,确定了自控罐发酵生产含鲑鱼生长激素酵母的工艺为含鱼生长因子酵母饲料添加剂的生长奠定了基础。     

基因工程菌发酵研究进展

基因工程菌发酵主要目标是获取高产外源基因表达蛋白。介绍并分析了基因工程菌发酵过程中表达系统、培养基、温度、pH值、溶解氧和诱导条件等因素对发酵的影响;论述了工程菌高密度培养所需的培养方式,并总结了基因工程菌发酵领域近年来的一些进展。     

重组人粒细胞集落刺激因子发酵工艺研究

通过对大肠杆菌（E.coli）表达重组人粒细胞集落刺激因子（rhG-CSF）工程菌DH5a的发酵工艺的研究,对影响工程菌生长和目标蛋白表达条件如：发酵培养基配方,pH值,诱导时间,分批补加营养物质等进行了优化。结果表明,采用优化的条件发酵后,工程菌得率达30g/L以上;目标蛋白表达量为30%以上。     

毕赤酵母工程菌高密度发酵研究与进展

毕赤酵母表达系统是近年来发展最为迅速的一种新型外源蛋白真核表达系统,被广泛应用于多种不同领域且成功表达了许多基因工程产品。高密度发酵技术已被广泛运用到毕赤酵母工程菌发酵工程当中。主要从毕赤酵母的表达常用菌株、载体及表型等方面介绍了其表达系统,从外源基因自身的特性、培养基的组成、温度、pH、溶氧量及补料流加策略方面阐述了对毕赤酵母高密度发酵的过程及蛋白质表达结果的影响,并对毕赤酵母工程菌高密度发酵进行了展望,为其今后的研究及应用提供借鉴。     

重组人白细胞介素-11工程菌的发酵条件研究

为了探讨发酵条件对大肠杆菌表达人白细胞介素-11融合蛋白的影响,利用正交实验设计,对工程菌的生长条件和人白细胞介素-11融合蛋白表达进行优化。在摇瓶中研究了培养基中的葡萄糖、蛋白胨、酵母抽提物的浓度、pH及摇床转速、装液量、接种量等。确定了工程菌生长及表达的培养基和培养条件:葡萄糖10g/L,蛋白胨20g/L,酵母抽提物10g/L,pH7.5,接种量10%,装液量10%,摇床转速220r/min及诱导时间为4~5h。然后在BiofloⅢ-5L发酵罐中以优化的发酵条件进行了3批实验,结果表明:工程菌量达到55g/L(DCW),重组人白细胞介素-11融合蛋白表达量为33%左右,为进行中试研究奠定了理论基础。     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.     

L-蛋氨酸酰化酶基因工程菌培养条件的研究

为提高L-氨基酰化酶基因工程菌1016的生物量及酶产量,优化了发酵工艺,确定了较佳的培养基成分,并初步考察了该酶的底物特异性。优化后工程菌摇瓶产酶稳定在980U/mL发酵液,发酵周期17 h,生物量A_(420)穗定在0.7。产酶量为原来的2倍以上。乙酰化的脂肪族氨基酸Met是该酶的最适底物。     

基因工程菌高密度发酵工艺研究进展

阐述了基因工程菌高密度发酵工艺的几个主要影响因素,包括重组菌构建、培养条件、生长抑制因子以及它们的控制技术。通过高密度发酵可以提高细胞生长密度、目的蛋白的表达含量。在高密度发酵过程中,会产生一些有害抑制代谢副产物,但通过分批补料可以降低影响。     

Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass

Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.     

Poly (glutamic acid)--an emerging biopolymer of commercial interest

Poly (γ-glutamic acid) (PGA) is water-soluble, anionic, biodegradable, and edible biopolymer produced by Bacillus subtilis. It has multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. The production of PGA has already been established on the industrial scale. Various studies regarding the fermentative production, downstream processing and characterization of PGA have been reported in the literature. This review provides updated information on fermentative production of PGA by various bacterial strains and effect of fermentation conditions and media component on production of PGA in submerged as well as solid state fermentation. Information on the application of genetic engineering for enhancement of yield of PGA, kinetic studies for production of PGA in submerged fermentation and recovery and purification of PGA is included. An attempt has also been made to review the current and potential applications of PGA. This review may contribute to further development of this commercially and academically interesting biopolymer.     

Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines

Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6 g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.     

毕赤酵母工程菌高密度发酵的研究进展

近年来毕赤酵母已成为一种优越的异源蛋白表达系统。而提高目的蛋白的表达水平,高密度发酵已成为关键技术环节之一。我们从毕赤酵母工程菌的选择、培养基的优化设计,以及发酵工程过程控制等方面简要阐述毕赤酵母的高密度发酵,并提出了工程菌在高密度发酵过程中存在的问题。     

p LA-PEDV-S1质粒拷贝数与发酵重组乳酸菌S1表达量之间的相关性研究

探究质粒拷贝数以及目标蛋白S1表达量与发酵时间的关系,从而确定放大生产p LA-PEDV-S1/Lactobacillus casei的最佳发酵时间。通过发酵重组干酪乳杆菌,绘制重组干酪乳杆菌的生长曲线,确定其生长的最佳时期。将p LA-PEDV-S1/L. casei分别接种至添加抗生素和不添加抗生素的MRS培养基中传代培养,进行稳定性实验。使用荧光定量PCR方法检测重组干酪乳杆菌中质粒的拷贝数,使用流式细胞术检测乳酸菌表达的目标蛋白。重组菌在7 h达到生长顶点,传至120代时外源质粒并无丢失情况出现。质粒拷贝数在9 h达到峰值29. 34,表达目标蛋白的重组菌在7 h达到峰值97. 98%。结果显示在细菌生长的对数生长期末期,质粒拷贝数最高且S1表达量最多;在平台期随着发酵时间的增加,质粒拷贝数逐渐降低,S1表达量也相应减少。发酵的最佳时间为7～10 h,质粒拷贝数与S1表达量之间存在着正相关性。     

牛肠激酶催化亚基工程菌培养条件的探索与鉴定

目的:探索牛肠激酶催化亚基工程菌高效表达的培养条件。方法:牛肠激酶催化亚基基因的初步表达,筛选构建工程菌,从以下方面入手优化培养条件:培养基的组成、摇瓶发酵培养条件、培养基的PH、种龄、接种量、培养时间等,并且通过Western Blotting和SDS-PAGE等鉴定不同条件下的实验结果,从而确定最佳培养条件。结果:通过鉴定实验结果,从不同种平行培养条件中选择产量最高的培养条件,作为最优培养条件。结论:成功地探索并鉴定了工程菌的适宜培养条件。