Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Maximizing recombinant human serum albumin production in a Muts Pichia pastoris strain

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Mut^s Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022–0.0024/h) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24 to 28^oC was effective to control the specific growth rate at low level (≤0.0024/h) during methanol induction phase while maintaining high specific productivity [0.0908 mg_rHSA/(g_wcw h)]. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1488–1496, 2014     

重组人骨保护素片段OPG179与人血清白蛋白在毕赤酵母中的融合表达

目的:在毕赤酵母中融合表达人骨保护素(OPG)片段OPG179与人血清白蛋白(HSA).方法:通过RT-PCR扩增获得OPG179基因,构建表达质粒pHILD2-rhOPG179-HSA,转化至毕赤酵母菌中进行表达、纯化,对分泌表达产物进行SDS-PAGE及Western印迹检测.结果:酶切鉴定与测序结果显示重组表达质...     

Expression of human serum albumin in metylotrophic yeast Pichia pastoris and its structural and functional analysis

The stable strain of methylotrophic yeast Pichia pastoris secreting human serum albumin into cultural medium was obtained. Optimal conditions for expression of the protein were determined. We characterized the recombinant protein by mass spectrometry and circular dichroism and analyzed its catalytic activity.     

Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris.

A novel recombinant exendin-4 human serum albumin fusion protein (rEx-4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex-4 is a 39-amino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma half-life (t(1/2) = 2.4 h). To extend the half-life of Ex-4 molecule in vivo, we designed a genetically engineered Ex-4/HSA fusion protein. Between Ex-4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically > 98%) by a three-step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex-4 for interaction with GLP-1 receptors in vitro. Results from oral glucose tolerance test indicated that rEx-4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx-4/HSA had a much longer plasma half-life. Therefore, rEx-4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy.     

人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达

目的：在巴斯德毕赤酵母中表达有降糖活性的人胰高血糖素样肽-1（hGLP-1）突变体（2Gly-hGLP-1）与人血清白蛋白（HSA）的融合蛋白。方法：为将GLP-1氨基酸序列第2位的丙氨酸（Ala）定点突变为甘氨酸（Gly）,根据毕赤酵母偏爱密码子合成编码2Gly-hGLP-1的基因;采用重叠PCR法拼接2Gly-hGLP-1和HSA的基因,使得2Gly-hGLP-1的C端与HSA的N端通过甘氨酸五肽接头连接;将该融合基因插入表达载体pPIC9构建为重组载体pPIC9/2Gly-hGLP-1-HSA,电击转化至毕赤酵母GS115细胞,通过表型筛选和诱导表达实验获得高效表达菌株;工程菌在5L发酵罐中培养后,对发酵产物进行分离纯化和生物学活性分析。结果：融合蛋白在5L发酵罐中的表达量约为200mg/L,经纯化后纯度可达95%以上;小鼠糖耐量实验表明该融合蛋白具有明显的控血糖活性。结论：在毕赤酵母中分泌表达的融合蛋白2Gly-hGLP-1-HSA具有降血糖活性。     

Expression of recombinant GFP-actin fusion protein in the methylotrophic yeast Pichia pastoris

The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.     

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.     

重组阿拉伯糖苷酶在毕赤酵母中的分泌表达及其活性分析

假密环菌是一种果树腐病菌,具有较强的分解代谢纤维素的能力。本文根据已克隆出的阿拉伯糖苷酶基因序列（Accession Number AJ620046）设计引物从假密环菌的mRNA中克隆出阿拉伯糖苷酶的ORF片段,构建重组质粒pPIC9-AF,与组氨酸标签融合,电转化毕赤酵母菌GS115并进行诱导表达。所得到的重组阿拉伯糖苷酶在30-35℃时有较高的酶活,最适反应活性pH值在6.0-8.0之间,而在pH4.0－8.0范围内酶活基本保持在80%以上,比大多已报道的阿拉伯糖苷酶最适pH范围宽。本研究工作对于高效表达重组阿拉伯糖苷酶以及对其进一步的酶学性质研究提供了一个良好的基础。     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.     

糖化酶、木聚糖酶融合蛋白在毕赤酵母中的高效表达

通过RT-PCR方法,以埃默森篮状菌(Talaromyces emersonii)总RNA为模板,克隆出糖化酶(glucoamylase,amyA)基因的成熟肽编码序列(1 857 bp),编码618个氨基酸;以类芽孢杆菌(Paenibacillus sp.)H10-3基因组DNA为模板,克隆出木聚糖酶(xylanaseA,xynA)基因的成熟肽编码序列(636 bp),编码211个氨基酸.通过重叠延伸PCR( SOE-PCR)得到拼接片段amyA-l-xynA,并将其克隆到毕赤酵母表达载体pPIC9中,得到重组质粒pPIC9-amyA-l-xynA,重组质粒线性化后经电击转化到毕赤酵母(Pichia pastoris)GS115中,得到了表达成功的工程菌ALX2.在ALX2发酵上清液中同时检测到糖化酶活性(10.7 U/mL)和木聚糖酶活性( 51.8 U/mL).     

Mutation of cysteine residues increases heterologous expression of peach expansin in the methylotrophic yeast Pichia pastoris

The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l⁻¹, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.     

人Tumstatin在毕赤酵母中的表达和活性分析

利用PCR技术从重组质粒pET-3c-tum中扩增人tumstatin的cDNA片段,连入pPICZαA酵母表达载体,获得的重组质粒pPICZα-tum电激法转化毕赤酵母GS115。经表型鉴定、诱导表达筛选,得到可分泌表达人tumstatin的重组酵母转化子,表达蛋白质的相对分子量约30kD,表达量约25mg/L。表达上清经超滤浓缩和离子交换法初步纯化,所得产物具有免疫活性,能够抑制内皮细胞增殖,诱导其发生细胞凋亡,并能抑制鸡胚尿囊膜血管生成。     

Production of recombinant human erythropoietin from Pichia pastoris and its structural analysis

AIMS: To design and investigate a recombinant expression system producing a therapeutically important glycoprotein, human erythropoietin (rHuEPO), by Pichia pastoris. METHODS AND RESULTS: EPO cDNA was cloned into pPICZalphaA for expression under control of AOX1 promoter and fused, on the amino-terminal end, with a polyhistidine tag for rapid purification. A target site for factor Xa protease was also introduced, such that cleavage in vitro produced a mature form of rHuEPO having the native N- and C-termini. RHuEPO was characterized as to the extent and nature of N-linked glycosylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and western blotting. The rHuEPO produced was approximately 30 kDa. All three N-linked glycosylation sites were occupied dominantly by Man(17)(GlcNAc)(2). N-glycanase-treated rHuEPO purified but not digested with factor-Xa-protease, showed a spectral peak centered about m/z 20400 Da. CONCLUSIONS: The native polypeptide form of human EPO (c. 18 kDa) was obtained for the first time in P. pastoris expression system, after affinity purification, deglycosylation and factor-Xa-protease digestion. The amount of sodium dodecyl sulfate used prior to deglycosylation was found to be crucial in determining the dominant form of glycan in glycoproteins. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel approaches to protein expression and purification system and structural analysis presented, would be important especially for therapeutic proteins expressed in P. pastoris.     

Expression of recombinant vitellogenin in the yeast Pichia pastoris

Vitellogenin (Vtg) plays vital roles as precursor to the yolk proteins and as carrier for lipids, carbohydrates, phosphates, metal ions, vitamins, and hormones into the oocytes during the massive deposition of yolk nutrients for subsequent nourishment of the developing embryos. Reproductive success is highly sensitive to the nutritional quality of the broodstock diet, which greatly affects the egg and larval viability. We present a novel strategy for genetically engineering a Pichia pastoris yeast strain that constitutively produces recombinant Vtg (rVtg), for application as an enriched feed. The tilapia Oreochromis aureus Vtg (OaVtg) cDNA (5.3 kb) was cloned into a nonsecretory pGAPZA vector. Clones containing up to 31 copies of glyceraldehyde-3-phosphate dehydrogenase (GAP)-promoter-driven Vtg expression cassettes were isolated. These clones expressed a membrane-associated intracellular rVtg protein of 194 kDa, constituting up to 1.16% of total protein. To facilitate future purification of rVtg, we explored the possibility of secreting rVtg using the native Vtg secretion signal and the alpha-factor secretion signal of Saccharomyces cerevisiae. However, neither signal promoted the secretion of rVtg. The clones maximally expressed rVtg at 23 degrees C, reaching a peak at 22 h in shake flasks and 16 h in a fermentor. The clones exhibited a significant increase in essential amino acids and long-chain polyunsaturated fatty acids, which are important for its application as a high-quality nutrient feed.     

重组HSA-hG-CSF融合蛋白在毕赤酵母中的表达

为了延长G-CSF半衰期,我们利用甲醇酵母表达重组人血清白蛋白融合的集落细胞刺激因子（rHSA-G-CSF）。用PCR方法从人胎肝cDNA文库扩增出HSA cDNA序列,hG-CSFcDNA序列从大肠表达载体中酶切获取。将HSA和hG-CSF两片段连接后,克隆到酵母分泌型表达载体pGENYK中,酶切线性化后原生质体转化导入酵母细胞进行整合。工程菌经发酵灌培养表达,层析法分离纯化融合蛋白。纯化的融合蛋白经Western 印迹分析表明具有HSA和G-CSF的免役原性,体外生物学活性分析表明,同縻尔数的融合表达产物的活性为E.coli表达G-CSF单体的活性的50%以上。体内动物实验研究表明,经HSA融合的G-CSF的半衰期为G-CSF单体的15-20倍。甲醇酵母表达的融合HSA的G-CSF具有比G-CSF更长的半衰期,有良好的临床应用前景。     

毕赤酵母醇氧化酶1启动子突变体的分离与鉴定

目的：通过醇氧化酶1启动子突变,筛选毕赤酵母高水平表达菌株。方法：通过致错PCR构建醇氧化酶1启动子突变体,经酶切连接到改造过的质粒HSA-pPIC9上,转化毕赤酵母GS115感受态细胞,摇瓶培养表达筛选人血清白蛋白（HSA）高表达突变体菌株。结果：克隆测序结果表明,突变的醇氧化酶1启动子-910和-569位点处共2个碱基发生了T→C突变;获得一株高表达菌株,摇瓶中HSA的表达量由200mg／L提高到335mg／L。结论：通过醇氧化酶1启动子突变成功构建了HSA高表达菌株。     

重组人白细胞介素21在毕赤酵母中的分泌表达及活性分析

为了获得有活性的重组人白细胞介素21(rhIL-21),本研究建立了在毕赤酵母(Pichia pastoris)中分泌表达rhIL-21的技术。首先,通过RT-PCR从人外周血淋巴细胞中扩增IL-21cDNA,克隆到酵母表达载体pPIC9K中,构建了重组酵母表达载体pPIC9K-hIL21 cDNA;然后,线性化的载体转化毕赤酵母表达菌株GS115,经抗性梯度筛选获得了多拷贝重组酵母菌;加入甲醇诱导培养后,SDS-PAGE和Western blotting检测到培养液中有rhIL-21的分泌表达,相对分子量约为16kD,ELISA结果显示摇瓶表达量可达229.28mg/L;表达上清经阳离子交换介质SPSepharose Fast Flow纯化后,目的蛋白纯度达到95%。细胞增殖试验结果显示该rhIL-21联合刀豆蛋白A(ConA)对人淋巴细胞的增殖具有显著的促进作用。本研究首次成功在毕赤酵母表达系统中分泌表达了有生物活性的rhIL-21,为相关疾病免疫治疗的研究奠定了基础。     

人血管内皮细胞生长抑制因子在巴斯德毕赤酵母中的分泌表达

血管内皮细胞生长抑制因子抑制肿瘤部位新生血管的形成,切断肿瘤细胞营养供应及废物排泄通道,抑制肿瘤细胞恶性增殖。将编码成熟的人血管内皮细胞生长抑制因子基因克隆到表达载体pPICZα中,电转化P.pastorisGS115菌株,抗生素ZeocinTM浓度梯度筛选高抗性转化子,PCR筛选阳性重组菌株。经表型鉴定后,用甲醇进行诱导表达,SDS-PAGE和Western印迹杂交结果证实了表达产物为重组人血管内皮细胞生长抑制因子-his6融合蛋白,表达量约为5mg/L。经细胞毒性试验测定,表达产物对人脐静脉内皮细胞HUVEC增殖具有较明显的抑制作用。     

人血清白蛋白在乳酸克鲁维酵母中的表达

以乳酸克鲁维酵母(Kluyveromyces lactis,K.lactis)GG799为宿主对人血清白蛋白(HSA)进行分泌表达。以pPIC9k-HSA为模板,采用带有XhoⅠ和NotⅠ酶切位点的引物PCR扩增获得HSA基因,经XhoⅠ和NotⅠ双酶切后插入pKLAC1,构建表达载体pKLAC1-HSA。经SalⅡ线性化后,电击转化K.lactis GG799,用含5 mmol/L乙酰胺的YCB平板筛选阳性转化子。提取基因组DNA,采用PCR方法对转化子鉴定后进行摇瓶发酵。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析发酵上清液中的表达产物,并初步分析酵母基础N源(YNB)对HSA在K.lactis GG799中表达的影响。结果表明,HSA成功在K.lactis GG799中分泌表达,表达量为81μg/mL,遗传稳定性好。