耐热羧肽酶Taq基因在毕赤酵母中的表达

为了获取表达羧肽酶Taq毕赤酵母工程菌,通过密码子优化,依据毕赤酵母密码子偏爱性,在体外合成了栖热水生菌的耐热羧肽酶Taq基因。将该基因克隆到毕赤酵母表达载体p HBM905A上并引入6×His标签,构建了重组质粒p HBM905A-Cpase Taq。将该重组质粒转化毕赤酵母GS115,经1%甲醇诱导表达72 h,酶产量达0.1 mg/m L。对纯化的重组酶进行酶活性分析表明在75℃,p H为7.5时,该酶比酶活性为80 U/mg。本研究首次证明了羧肽酶Taq能在毕赤酵母中有效分泌表达,可以被大量制备,进而为多肽水解为氨基酸奠定工业基础。     

金针菇免疫调节蛋白基因fip-fve在毕赤酵母GS115中诱导型和组成型表达

为获得稳定来源并且具有生物学活性的重组金针菇免疫调节蛋白(Fip-fve),将fip-fve基因转至毕赤酵母GS115中进行诱导型和组成型表达。用PCR方法从金针菇子实体基因组DNA中扩增fip-fve基因,连接至pPIC9构建诱导型表达载体pPIC9-FIP-fve,从毕赤酵母基因组DNA中扩增三磷酸甘油醛脱氢酶启动子(pgap),替换pPIC9-FIP-fve上乙醇氧化酶启动子(paox1)构建组成型表达载体pPIC9-PGAP-FIP-fve。将线性化的两种表达载体用PEG法转化毕赤酵母GS115,经组氨酸缺失培养基筛选和酵母菌落PCR鉴定后进行表达。结果表明,重组Fip-fve在以甲醇(1%,V/V)为碳源进行诱导型表达4 d达到最高,粗蛋白表达量为158.2 mg/L,在以葡萄糖(10%)和甘油(1%,V/V)为碳源进行组成型分别在表达第4天和第5天达到最高,粗蛋白分别为46.3 mg/L和29.5 mg/L。SDS-PAGE及Western blotting证明重组Fip-fve已正确表达,血细胞凝集活性检测初步证明重组Fip-fve具有良好生物学活性。     

不同碳源下毕赤酵母GS115蛋白组学分析

毕赤酵母(Pichia pastoris)表达系统是目前最为成功的外源蛋白表达系统之一。该表达系统不存在原核表达系统的内毒素难以除去的问题,也不存在哺乳动物细胞表达系统的病毒和支原体污染问题;并能够对目的蛋白进行类似于高等真核生物的信号肽剪切、二硫键形成、糖基化等蛋白翻译后加工。但是不论是胞内表达或是分泌表达,大多数外源蛋白均面临着被降解的问题,这也是影响目的蛋白表达量的一个重要因素,同时还增加了纯化目的蛋白的难度。目的:研究毕赤酵母GS115在不同碳源培养过程中胞内外蛋白质组学的差异,指导毕赤酵母表达系统的优化。方法:利用LC-ESI-MS/MS方法分析了不同碳源的四种培养基中毕赤酵母GS115的胞内和胞外蛋白种类,利用Griffin等的计算方法计算各个蛋白的含量。结果:利用LC-ESI-MS/MS结合Griffin等的归一化非标定量法SI_N得到GS115胞内胞外详尽的蛋白质种类及准确百分比含量。结论:分析不同培养基之间蛋白质组成的差异,从而为以后构建新的毕赤酵母表达体系,为外源蛋白表达系统的优化提供一定的指导意义。     

Synthesis and high expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115

A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.     

小鼠B16黑色素瘤细胞核迁移蛋白基因的克隆及其在毕赤酵母中的表达

利用RT-PCR方法从小鼠B16黑色素瘤细胞中克隆了全长核迁移蛋白C(Nuclear distribution C,NUDC)基因,构建了该基因的毕赤酵母表达质粒pPICZaA-NUDC并在受体细胞Pichia pastoris GS115中获得表达,通过制备融合表达蛋白的多克隆抗体分析其对黑色素瘤细胞活性的影响.结果表明,克隆的NUDC基因全长1 000bp,含有完整的编码框,经SDS-PAGE和Western blotting分析表明,该基因相对分子质量约为45 kD.表达的蛋白能与兔抗NUDC抗体发生反应,制备的NUDC蛋白抗体可抑制黑色素瘤细胞生长.     

黑曲霉果胶裂解酶基因毕赤酵母在Pichia pastoris GS115中的表达

利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.     

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ?¹²-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ?¹⁵-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ?⁹-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.     

Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau

It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.     

用GAP启动子在Pichia pastoris GS115中组成型表达鼠灰链霉菌腺苷酸脱氨酶

【目的】构建产AMP脱氨酶的重组毕赤酵母(Pichia pastoris GS115)菌株,并初步优化其发酵条件。【方法】以鼠灰链霉菌(Streptomyces murinus)基因组为模板PCR扩增获得腺苷酸脱氨酶基因AMPD,以pGAP9K为载体构建重组表达质粒pGAP9K-AMPD并通过电转化法转入Pichia pastoris GS115,筛选转化子对其酶活进行测定,并初步优化其发酵条件。【结果】构建了毕赤酵母重组菌,通过分光光度法测定,显示重组菌有明显的酶活;初步优化发酵条件为:该重组菌最适发酵培养基为:甘油2%,蛋白胨2%,酵母膏1%,KH2PO40.5%,MgSO4·7H2O0.05%,pH 6.0;发酵条件为:接种龄24 h,转接量3%,30°C﹑200 r/min培养96 h,取发酵上清液测定酶活,重组菌腺苷酸脱氨酶酶活达到2 230±60 U/mL。【结论】构建了一株产AMP脱氨酶活性较高的重组毕赤酵母菌株,并通过优化发酵条件使其酶活达到2 230±60 U/mL。为AMP脱氨酶工业化生产奠定了一定的基础。     

香菇菌C91-3凋亡相关蛋白24414在毕赤酵母GS115中的表达与鉴定

目的通过构建毕赤酵母表达载体将香菇菌C91-3凋亡相关蛋白24414在毕赤酵母GS115中进行表达,同时对表达产物进行鉴定。方法从香菇菌C91-3菌丝体中提取总RNA,根据转录组测序结果,用3＇-Full RACE、5＇-Full RACE方法获得24414基因,并将其克隆到毕赤酵母的表达载体pPIC9K中,构建真核重组表达质粒pPIC9K-24414。用电转化的方法将此质粒转化到毕赤酵母GS115中并进行诱导表达,对表达产物用Westen-blot方法进行鉴定。结果通过菌落PCR和基因序列分析确定插入pPIC9K中的片段为24414基因片段,通过Westen-blot方法确定所表达蛋白为目的蛋白。结论重组质粒pPIC9K-24414成功构建,目的凋亡相关蛋白24414在毕赤酵母GS115中成功表达,为进一步研究香菇菌C91-3凋亡相关蛋白24414的生物学功能奠定了基础。     

Fast development of Pichia pastoris GS115 Mut+ cultures employing batch-to-batch control and hybrid semi-parametric modeling

In this paper, we implemented a model-based optimization platform for fast development of Pichia pastoris cultures employing batch-to-batch control and hybrid semi-parametric modeling. We illustrate the methodology with a P. pastoris GS115 strain expressing a single-chain antibody fragment (scFv) by determining the optimal time profiles of temperature, pH, glycerol feeding and methanol feeding that maximize the endpoint scFv titer. The first hybrid model was identified from data of six exploratory experiments carried out in a pilot 50-L reactor. This model was subsequently used to maximize the final scFv titer of the proceeding batch employing a dynamic optimization program. Thereupon, the optimized time profiles of control variables were implemented in the pilot reactor and the resulting new data set was used to re-identify the hybrid model and to re-optimize the next batch. The iterative batch-to-batch optimization was stopped after 4 complete optimized batches with the final scFv titer stabilizing at 49.5 mg/L. In relation to the baseline batch (executed according to the Pichia fermentation guidelines by Invitrogen) a more than fourfold increase in scFv titer was achieved. The biomass concentration at induction and the methanol feeding rate profile were found to be the most critical control degrees of freedom to maximize scFv titer.     

Effect of disruption of Pichia pastoris YPS1 gene on viability and production of recombinant proteins

In the present study, we constructed a Pichia pastoris mutant strain PS111 that lacks one member of the yapsin family through disruption of the YPS1 gene coding for aspartic protease yapsin 1. Under normal growth conditions, the PS111 mutant strain did not show detectable growth defects. Unlike the S. cerevisiae yps1 mutant, the P. pastoris PS111 strain showed no sensitivity when grown in the presence of CaCl₂, elevated temperature (37°C), under acid (pH 4.9) and alkaline (pH 8.3) conditions. Unlike the S. cerevisiae, the P. pastoris yps1 mutant showed decreased growth phenotype induced by cell wall-perturbing reagent sodium dodecyl sulfate (SDS) only when the concentration of SDS was increased by ten times. The use of the yps1 disruptant to produce human interferon alpha16 (hINF-α16) prevents proteolysis, which occurs in the wildtype strain. It was found that the degradation of recombinant protein Alburon composed of human serum albumin (HSA) and hINF-α16 was slightly decreased in the strain lacking yapsin 1.     

Expression, purification, and bioactivity of (GLP-1A2G)2-HSA analogs in Pichia pastoris GS115

We developed (GLP-1_A2G)₂-HSA (GGH) analogs that are resistant to degradation and also show high serum glucose-reducing activity in vivo. Five analogs with N-terminal extensions were designed based on the protein GGH. Next, we constructed recombinant plasmids capable of expressing the five analogs in methylotrophic yeast Pichia pastoris GS115. Expression reached 150 mg/L in a small-scale incubation. Fusion proteins were successfully purified from the supernatant using ultrafiltration concentration, affinity absorption chromatography, hydrophobic chromatography, ion exchange chromatography and gel filtration. A single band was observed on SDS-PAGE and the purity was 97%. Activity test results suggested that both A-GGH and G-GGH showed better activity in vitro and that their cAMP levels were significantly increased by 10-fold compared to GGH without N-terminal extension. Additionally, A-GGH efficiently enhanced the glucoselowering effect, which was maintained after the administration for 24 h. A-GGH is a potential drug for treating type 2 diabetes.     

苦瓜MAP30基因的毕赤酵母表达载体构建及重组菌株的筛选

目的:从苦瓜中克隆MAP30全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法。方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异性的引物,通过PCR技术扩增出全长861bp的MAP30基因。该基因经XhoⅠ和EcoRⅠ双酶切,连接至毕赤酵母表达载体pPIC9中。重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株。结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核苷酸同源性达99.9%,氨基酸同源性达100%。菌落PCR显示外源基因已整合入酵母GS115菌株中。结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础。     

应用双质粒共表达体系提高融合蛋白GGH在毕赤酵母GS115中的表达量

为实现人胰高血糖素样肽-1-人血清白蛋白融合蛋白 ((GLP-1A2G)2-HSA,简称GGH) 的规模化制备,通过pPICZαB与pPIC9K双质粒共表达体系提高融合蛋白GGH在毕赤酵母中的表达量。首先运用PCR技术扩增出融合蛋白GGH的基因片段,构建了表达质粒pPICZαB-ggh,并电转至经载体pPIC9K-ggh异位整合的GGH分泌型菌株——毕赤酵母GS115/F2;然后采用免疫学方法并结合高浓度抗生素筛选获得高产菌GS115/F3,在30 ℃,3％甲醇诱导80 h后GGH的表达量达到了491 m     

Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115

A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7-380 mL columns and the process was performed at the pilot scale using 0.5-3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01g of rBoNTE(Hc)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50's of BoNT/E.     

Development of an enzymatic synthesis approach to produce phloridzin using Malus x domestica glycosyltransferase in engineered Pichia pastoris GS115

Phloridzin has been an attractive target for the development of preparation methodologies due to its various applications as pharmaceutical ingredient and food additive. In this study, an enzymatic synthesis approach using engineered P. pastoris GS115 was developed to produce phloridzin. Four factors, identifying as key factors affecting the phloridzin yield, were optimized by response surface methodology as follows: induction time 121.8 h, pH 7.0, methanol 0.75%, and a rotation speed 194 rpm. The maximal yield of phloridzin reached 41.59 mg/L (95.31 μM) and the specific bioconversion rate of substrate was up to 98.50% with 1L working volume in a 5L bioreactor under the optimal conditions. Phloridzin with purity of 93.98% and recovery of 78.14% was achieved just using affinity chromatography and preparative high performance liquid chromatography (Pre-HPLC). These results confirm the enzymatic synthesis developed provides an efficient alternative to traditional approach for the preparation of phloridzin in vitro.