诱导相温度对毕赤酵母表达重组人复合α干扰素聚合的影响

研究了Pichia pastoris表达重组人复合α干扰素(cIFN)时诱导期温度对cIFN形成聚合体的影响。在5L罐上考察毕赤酵母在不同温度(30、25、20℃)下诱导时菌体生长和cIFN表达的差异,发现毕赤酵母在20℃下菌体生长不受影响,总蛋白表达量有所降低,相当于30℃发酵时胞外总蛋白的67.8%。但是通过非还原性电泳、Native-PAGE及Western blotting分析发现较高温度(30℃)诱导表达时,分泌到胞外的cIFN容易形成聚合体,单体含量很少。诱导相控制温度为20℃时,明显降低了cIFN聚合体的形成,cIFN单体量为570mg/L,发酵上清液抗病毒活性为1.05×109IU/mL。与控制诱导相温度为30℃相比,cIFN单体量提高了7.2倍,发酵上清液中单位体积的cIFN抗病毒活性提高了38.7倍。     

pH对毕赤酵母表达重组人复合α干扰素的降解影响

使用Pichiapastoris表达重组人复合α干扰素(cIFN)会发生降解、聚合等不均一表达的现象.在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响,结果发现在适合酵母生长的pH 3.0～7.0范围内,当诱导pH为4.0～5.0时,cIFN不均一表达现象最少,生物活性达到2.5×108 IU/mL.通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因:发现低诱导pH下细胞死亡率升高释放更多酶系,高诱导pH下蛋白酶活性明显增大,两者都使蛋白酶作用加强,加剧cIFN的降解;特别是诱导pH为7.0时,适宜的pH使蛋白酶酶活陡升,将cIFN完全降解.     

pH对毕赤酵母表达重组人复合a干扰素的降解影响

使用Pichia pastoris表达重组人复合a干扰素(cIFN)会发生降解、聚合等不均一表达的现象。在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响, 结果发现在适合酵母生长的pH 3.0~7.0范围内, 当诱导pH为4.0~5.0时, cIFN不均一表达现象最少, 生物活性达到2.5×108 IU/mL。通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因：发现低诱导pH下细胞死亡率升高释放更多酶系, 高诱导pH下蛋白酶活性明显增大, 两者都使蛋白酶作用加强, 加剧cIFN的降解; 特别是诱导pH为7.0时, 适宜的pH使蛋白酶酶活陡升, 将cIFN完全降解。     

pH对毕赤酵母表达重组人复合a干扰素的降解影响

使用Pichia pastoris表达重组人复合a干扰素(cIFN)会发生降解、聚合等不均一表达的现象。在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响, 结果发现在适合酵母生长的pH 3.0~7.0范围内, 当诱导pH为4.0~5.0时, cIFN不均一表达现象最少, 生物活性达到2.5×108 IU/mL。通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因：发现低诱导pH下细胞死亡率升高释放更多酶系, 高诱导pH下蛋白酶活性明显增大, 两者都使蛋白酶作用加强, 加剧cIFN的降解; 特别是诱导pH为7.0时, 适宜的pH使蛋白酶酶活陡升, 将cIFN完全降解。     

不同控制模式对毕赤酵母表达IL-2-HSA的比较研究

以溶氧（DO）和甲醇浓度作为控制指标,分别考察了诱导表达阶段DO和甲醇浓度对毕赤酵母高密度发酵表达白介素-2-白蛋白融合蛋白（IL-2-HSA）的影响。通过实验比较得出：诱导阶段控制DO对IL-2-HSA表达影响不明显,而甲醇浓度对IL-2-HSA影响很大。高甲醇浓度（15～25g／L）有利于IL-2-HSA的表达,发酵结束时的表达量约是低甲醇浓度（5～8g／L）诱导下的2倍。此外,在诱导阶段,通过限制性添加甘油可以有效提高菌体的呼吸活性,促进菌体生长。     

甲醇-山梨醇混合碳源诱导提高抗HER2抗体在糖基工程毕赤酵母中的表达

目的：以抗HER2抗体为模型,研究抗体在糖基工程酵母菌中的表达及工程菌发酵技术。方法：首先通过摇瓶试验分析诱导用甲醇浓度对抗体表达的影响,并用高表达HER2的SK-BR-3细胞分析抗HER2抗体的抗原结合活性。以此为基础,在5 L发酵罐中研究甲醇-山梨醇混合碳源流加诱导对抗HER2抗体表达水平的影响;收集发酵培养液,采用阳离子交换层析对目标产物进行纯化;利用SDS-PAGE、Western印迹、Lowry法对抗体的相对分子质量、浓度等进行分析。结果：摇瓶试验结果表明,甲醇浓度为0.5%时抗体表达量最高,且糖基工程毕赤酵母菌表达的抗HER2抗体具有与SK-BR-3细胞抗原结合的活性;在5 L发酵罐中,利用甲醇和山梨醇混合诱导方式发酵表达抗体,其表达量可提高至0.6 g/L,比摇瓶诱导表达的抗体产量提高了近10倍;非还原SDS-PAGE及Western印迹表明抗体相对分子质量为1.5×105,与商业化抗体Herceptin的大小一致;经过一步阳离子交换层析纯化,纯化后抗体浓度为0.365 g/L。结论：采用甲醇-山梨醇混合碳源诱导方式在5 L发酵罐中进行发酵表达,能够提高抗HER2抗体在糖基工程酵母菌中的表达量,本研究可为抗体在酵母中的规模发酵技术提供重要参考。     

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。     

利用Pichia pastoris生产S-腺苷甲硫氨酸的发酵工艺

在摇瓶中考察了重组Pichia pastoris发酵的诱导剂量,L-甲硫氨酸,以及pH对腺苷甲硫氨酸产量的影响.放大到3.7 L发酵罐和30 L发酵罐后,研究了重组细胞的发酵过程变化,对S-腺苷甲硫氨酸初步纯化.摇瓶中优化后的发酵条件是:每天添加1%甲醇诱导,L-甲硫氨酸为50mmol/L,培养基pH 5.0.培养144 h后SAM产量达到2.32 g/L.3.7 L发酵罐中发酵251 h后细胞浓度为120 g/L,SAM总量为15.18 g.放大到30 L发酵罐中,发酵225.5 h后细胞浓度约为120 g/L,SAM总量为145.05 g.纯化后SAM的纯度为93.5%,回收率为84.5%.     

基于密码子优化的蛋白酶K在毕赤酵母中的表达及分离纯化

【目的】提高蛋白酶K在毕赤酵母中的表达产量,建立分离纯化方法。【方法】首先对蛋白酶K密码子进行优化,将其导入毕赤酵母GS115中实现分泌表达。然后对甲醇浓度、发酵温度和p H等表达条件进行优化,再对硫酸铵沉淀、亲和层析等纯化工艺进行比对分析。【结果】蛋白酶K密码子优化后实现了在毕赤酵母中的高效表达。在甲醇量0.75%、温度25°C和p H 7.0条件下进行发酵罐培养,蛋白酶K表达量达到2.2 g/L。采用Ni-NTA亲和柱对发酵液进行纯化可以得到较好的纯化效果。【结论】密码子优化后的蛋白酶K在毕赤酵母中高效表达并可以利用Ni-NTA亲和柱进行有效分离纯化。     

通过在低细胞浓度下启动甲醇诱导、优化碳/能量代谢模式促进毕赤酵母表达Monellin

重组毕赤酵母生产表达外源蛋白的过程中,一般在细胞达到高密度后开始启动甲醇诱导。也有报道指出,在较低细胞浓度下,启动甲醇诱导可以有效地控制整个发酵过程的溶解氧浓度,缓解毒副产物的积累,促进目标蛋白的表达。但是,该操作策略下,甲醇/能量调控机制不明,相关研究报道很少。文中以生产表达monellin(甜味蛋白)的重组毕赤酵母为模式菌株,通过在线分析计量甲醇消耗速率、CO2释放速率和O2摄取速率,探讨了不同细胞浓度下启动甲醇诱导和外源蛋白表达体系的甲醇/能量代谢模式。结果表明,在较低细胞浓度(50 g DCW/L)启动诱导并将温度控制在30℃,走向合成monellin前体物质途径的碳流最大(65%),且能与用于ATP再生的碳流形成最佳匹配;monellin的比合成速率与细胞比生长速率完全耦联,且耦联系数最大,比生长速率也较高;理论NADH(能量)利用效率η最高,η在甲醇诱导的绝大部分时段(89%)处于高水平(≥0.8),可以为monellin合成提供足够的能量。因此,该操作条件下,monellin浓度达到2.62 g/L的最高水平,是高细胞密度(100 g DCW/L)启动诱导策略下monellin浓度的2.5-4.9倍。     

Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is a powerful system for production of recombinant proteins, showing high ability to secrete properly folded proteins. A major plus is the strong AOX1 promoter highly induced by methanol. During growth on methanol, however, oxygen readily becomes limiting. In oxygen-limited cultivations of recombinant Pichia pastoris, the methanol concentration had a strong impact on the production of a single-chain antibody fragment (scFv). High methanol concentrations were required to compensate the lack of oxygen and fully induce recombinant protein production, at the same time reducing gratuitous biomass formation due to a lower biomass yield. Product concentrations of 60, 150, and 350 mg/L were obtained with methanol concentrations of 0.3, 1, and 3% (v/v). Moreover, accumulation of a putative product fragment that cannot be removed during affinity purification was prevented at high methanol concentrations. Cell vitality after 100 h was maintained above 98% and 96% of the culture with 0.3% and 3% methanol, respectively. In cultivations supplemented with oxygen, in contrast, methanol concentration between 0.3% and 3% did not influence the product yield of 300-400 mg/L. Thus, efficient recombinant protein production under oxygen-limitation seems to require high methanol concentrations, enabling product concentration as high as otherwise obtained only with expensive supply of pure oxygen.     

Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-α mutant by Pichia pastoris

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-α mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of β-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.     

The inhibition of aggregation of recombinant human consensus interferon-α mutant during <Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentation

Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-α mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20°C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20°C was 4.04 times as high as that at 30°C. There was no obvious effect on the cell growth at 20°C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l⁻¹ Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20°C was 19.9-fold higher than that without Tween-20 at 30°C.     

Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis

Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G+C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K-zbtb7a-his(6) was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS-PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.     

Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion

In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75 mlmin(-1) per 10l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400 rpm. By increasing the anti-foam reagent to 0.6 mll(-1), the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120 mgl(-1) at 163 h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.     

重组人小分子抗体ScFv-Fc发酵条件的优化及纯化

目的：研究重组人小分子抗体ScFv—Fc在毕赤酵母中分泌表达的最佳条件,以及ScFv—Fc的纯化方法。方法：分别从甲醇浓度、pH、诱导时间等方面对毕赤酵母重组菌株产生ScFv-Fc的发酵过程进行了优化;通过硫酸铵沉淀结合proteinA亲和层析柱,对ScFv—Fc的纯化方法进行了研究。结果：确定ScFv—Fc在毕赤酵母中分泌表达的最佳条件为：在pH5．2的条件下,以0．5％甲醇诱导72h。经过proteinA亲和层析柱纯化后,ScFv—Fc纯度可达94％以上。结论：确定了ScFv-Fe在毕赤酵母中分泌表达的最佳条件以及纯化方法,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。     

Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris

The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.     

Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts

The coding sequence of the mature dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacterial chaperonin GroESL. However, the enzyme had no activity. Recombinant expression in Pichia pastoris and Saccharomyces cerevisiae yielded an active enzyme. Dalcochinase was expressed under methanol induction in P. pastoris, since this was much more efficient than constitutive expression in P. pastoris or in S. cerevisiae. Addition of 0.5% casamino acids to the culture medium stabilized the pH of the culture and increased the protein yield by 3- to 5-folds. Insertion of a polyhistidine-tag either after the N-terminal alpha factor signal sequence or at the C-terminus failed to assist in purification by immobilized metal-ion affinity chromatography (IMAC) due to post-translational processing at both termini. A new construct of dalcochinase with an N-terminal truncation following the propeptide and eight histidine residues enabled its purification by IMAC, following hydrophobic interaction chromatography. The purified recombinant dalcochinase was apparently composed of differently post-translationally modified forms, but had kinetic properties and pH and temperature optima comparable to natural dalcochinase. The procedures reported here overcome the limitation in enzyme supply from natural sources, and allow further studies on structure-function relationships in this enzyme.