Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.     

Properties of endogenous β-glucosidase of a Pichia anomala strain isolated from Sicilian musts and wines

The AL 112 strain, isolated from 361 yeast strains in Sicilian musts and wines, has been identified by biochemical and molecular methods as belonging to Pichia anomala, and your endogenous β-glucosidase (βG, EC 3.2.1.21) subsequently characterised. This strain not only has extremely high specific productivity of βG, but above all shows arabinosidase (Ara, EC 3.2.1.55) activity, essential for aroma enhancement of wine. βG from Al 112 is activated by ethanol at the concentrations typically found in wine; it is not inhibited by fructose, whilst glucose, a non-competitive inhibitor, despite lowering activity, actually protects the enzyme from factors that could damage it. It has an optimum temperature of 20 °C, compatible with typical cellar conditions, and stability in model must-wine and wine solutions ≥40 days.     

Expression of recombinant rabbit tissue factor in Pichia pastoris,and its application in a prothrombin time reagent

Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.     

东方肉座菌EU7-22纤维素内切酶Ⅰ的异源表达

[目的]实现东方肉座菌纤维素内切酶EGⅠ在毕赤酵母中的表达,获得重组EGⅠ。[方法]通过RT-PCR获得EGⅠ开放阅读框。将EGⅠ成熟肽和PHO1信号肽的DNA片段插入p PIC3. 5K后,重组表达载体电转化毕赤酵母。通过甲醇诱导表达和镍柱纯化获得EGⅠ。以羟甲基纤维素钠检测活性,以肽N-糖苷酶F分析N-糖基化,以SDSPAGE分析表达情况和糖基化修饰。[结果]获得EGⅠ分泌表达菌株,诱导96 h后上清液活性为0. 513±0. 002 U/m L,纯化后的EGⅠ活性为0. 558±0. 012 U/mg。SDS-PAGE表明EGⅠ分子量在100～180 k Da,远高于预测值47. 3k Da,经肽N-糖苷酶F处理后,降至63～75 k Da。[结论]实现了EGⅠ的分泌表达,获得活性为0. 558±0. 012 U/mg的糖基化重组EGⅠ。     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

奶牛α干扰素在毕赤酵母中的分泌表达及其生物活性测定

提取经新城疫病毒诱导培养的奶牛外周血淋巴细胞总RNA,应用RT-PCR方法扩增出奶牛α干扰素成熟蛋白基因,然后将特异性片段连接到pMD18-T载体,测序结果表明,扩增片段为牛α干扰素成熟蛋白序列,与Genebank上发表的α1干扰素序列同源性为98%。然后将特异性片段连接到含分泌信号肽序列的Pichiapastoris表达载体pPICZαA上,将重组质粒经SacI酶切线性化后电转化导入毕赤酵母菌株X-33。转化子经PCR分析鉴定后利用甘油增菌和甲醇诱导,实现了BoIFN-α在毕赤酵母系统中的分泌表达。SDS-PAGE结果显示表达产物分子量约为23kDa,比其推导结果(20kDa)略大,推测可能是因为表达产物发生了一定程度的糖基化。细胞病变抑制法结果表明,重组牛IFN-α具有较高的干扰素活性,约为4.1×10⁵U/ml,经微量蛋白测量仪测得,其表达量约为80μg/ml,比活为5.1×10⁶U/mg。     

超嗜热酯酶EST2在不同宿主中的异源高效表达研究

来源于超嗜热古菌Alicyclobacillus acidocaldarius的酯酶EST2是目前报道的活性最高的超嗜热酯酶,具有极大的工业应用价值。为促进EST2的生产应用,将其分别在大肠杆菌及毕赤酵母中进行异源表达,并就不同宿主对表达情况和重组酶酶学性质的影响进行了分析。在大肠杆菌和毕赤酵母中重组表达的EST2酶学性质基本一致:最适温度分别为75℃和77.5℃,最适pH均为8.0,比活力分别为4656.6 U/mg和4078.3 U/mg,70℃水浴保温4.5 h,残余活力均在70%以上。在摇瓶发酵的基础上,于5 L发酵罐中进行了重组大肠杆菌及毕赤酵母的高密度发酵。毕赤酵母高密度发酵120 h菌体干重达68 g/L,最大表达酶活力为959.6 U/ml。大肠杆菌高密度发酵25 h菌体干重达60.8 g/L,最大酶活力14825.6 U/ml,表达量是毕赤酵母的15.4倍,单位时间产量是酵母的74.2倍。结果表明大肠杆菌发酵周期短、表达量高,更适合进行嗜热酯酶EST2的高效生产,这为促进嗜热酯酶在工业生物技术产业的应用奠定了基础。     

毕赤酵母工程菌表达乙肝表面抗原结合蛋白(SBP)的发酵纯化工艺研究

乙肝表面抗原结合蛋白(HBsAg binding protein,SBP)是以HBsAg为探针,通过人肝cDNA噬菌体表达库筛选的一种人源蛋白。SBP可特异性结合乙肝表面抗原(Hepatitis B virus surface antigen, HBsAg),增强乙肝疫苗的免疫效果,是一种潜在快速高效的免疫佐剂。成功构建了分泌型高表达SBP的毕赤酵母工程菌。对该菌株进行了放大规模发酵表达,并对纯化工艺进行了研究。在发酵实时检测过程中,自诱导剂甲醇加入开始,SBP蛋白的分泌表达量随时间推移而逐渐增加,发现于38h达到最佳水平且此时杂蛋白含量最少,是放罐收集菌液的最佳时间。18L发酵液在低温离心除菌体和沉淀物后可得到15L的上清液,再利用截留量为5kDa的超滤膜包将上清液浓缩至2L,将浓缩后的上清液依次通过S200分子筛柱和TDEAE阴离子交换柱进行分离纯化,可得到300ml浓度为1.125mg/ml、纯度达98%的目标蛋白液,发酵液得率为22.5mg/L;最后将蛋白液定量分装冻干低温保存。所获得的放大规模SBP发酵诱导表达条件和SBP蛋白的分离纯化工艺,为SBP蛋白大规模生产奠定了坚实的基础。