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.     

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.     

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。     

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.     

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.     

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。     

甘油相控制策略对重组毕赤酵母表达瑞替普酶(reteplase)的影响

研究了甘油补料策略对毕赤酵母表达reteplase(rPA)的发酵过程中细胞的生长和rPA表达的影响。通过将对甘油补料速率由10g/L.h-1增大到20g/L.h-1,细胞比生长速率,甘油的比消耗速率,甘油得率等都得到极大提高。而且,诱导期细胞生长,甲醇消耗和rPA的生成速率都增加,rPA的最大表达量从140.2mg/L增加到199.5mg/L。此另外,甘油补料时间也是影响rPA表达的重要因素,甘油补料时间短,细胞密度小,表达rPA少,甘油补料时间长,细胞密度增大,rPA的表达速率也增加,但是到诱导后期,细胞死亡率增大,蛋白酶释放增加,rPA的降解增强。     

巴斯德毕赤酵母表达外源蛋白的优化策略

巴斯德毕赤酵母(Pichia pastoris)表达系统已成为外源蛋白最理想的表达系统之一,诸多的优点体现了其广泛的研究价值和应用价值。综述了P.pastoris表达外源蛋白时在载体选择与利用、外源基因改造、翻译后修饰及表达稳定性等方面的优化策略,以加速其应用。     

巴斯德毕赤酵母表达系统及其高水平表达策略

毕赤酵母表达系统是目前最为成功的外源蛋白表达系统之一,与现有的其它表达系统相比,巴斯德毕赤酵母在表达产物的加工、外分泌、翻译后修饰以及糖基化修饰等方面有明显的优势,现已广泛用于外源蛋白的表达。该文综述了其自身的优点,表达载体和宿主菌的特点,以及高水平表达外源基因的策略。     

猪白细胞介素-6在巴斯德毕氏酵母(Pichia pastoris)中的分泌表达

白细胞介素6 (Interleukin_6 ,IL_6 )是一种具有多种生物学效应的细胞因子,在疾病诊断与疫苗佐剂领域有广阔的应用前景。在本试验中,猪白细胞介素_6 (pIL_6 )的cDNA序列被克隆入甲醇酵母(Pichiapastoris)分泌表达载体pPIC9K中,并转化入P .pastorisGS115菌株。其重组菌株GS115 pPIC9K_IL6经1%甲醇诱导后,能分泌表达分子量约为2 4 5KD的重组蛋白,Westernblot确证为pIL_6。该酵母表达产物无N端糖基化修饰。用依赖IL6生长的B9细胞株检测提纯后的pIL_6 ,其生物学活性可达8×10 4 IU mg。     

重组人白细胞介素11在毕氏酵母中的表达

将人白细胞介素11基因选用酵母偏爱密码子人工合成全基因,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,G418筛选得到多拷贝转化子,甲醇诱导表达,纯化制备产物,经过SDS－PAGE、Western印迹及体内外生物学活性等分析表明,产物活性与E.coli融合表达的Neumega一致。     

牛促卵泡激素在毕赤酵母中的表达

目的:实现重组牛促卵泡激素在毕赤酵母中的表达。方法:依据毕赤酵母的密码子偏爱性设计并利用PCR方法合成了牛促卵泡激素的α亚基和带有6×his-tag的β亚基相应的DNA序列,构建表达载体pHIL-S-bFSH,转化毕赤酵母,表达蛋白进行ELISA定量和Western blot鉴定。结果:重组牛促卵泡激素的表达量为0.26mg/L,Western blot鉴定分子量为35kDa。结论:在毕赤酵母中实现了具有免疫源活性的重组牛促卵泡激素的表达。     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

人胰岛素在甲醇酵母Pichia pastoris中的分泌表达

将猪胰岛素前体基因和在其５’端引入９个氨基酸的间隔肽序列的ＰＩＰ基因插入到Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ的分泌表达质粒ｐＰＩＣ９中,得到分泌表达质粒ｐｐＩＣ９／ＰＩＰ和ｐＰＣ９／ｓｐ－ＰＩＰ７并用以转化ｐａｓｔｏｒｉｓ ＧＳ１１５。用点杂交筛选,获得高拷贝转化Ｐ３９（－ｓｐ）和Ｓ５１（＋ｓｐ）。     

人可溶性晚期糖基化终产物受体的酵母表达及其生物活性分析

将人可溶性晚期糖基化终产物受体(human soluble Receptor for advanced glycation end product,hsRAGE)cDNA插入巴斯德毕赤酵母组成型表达载体pGAPZαA,构建重组表达载体pGAPZαA-hsRAGE,并转化到毕赤酵母X-33中,利用含抗生素Zeocin的YPD平板筛选重组子。重组菌株在甘油醛-3-磷酸脱氢酶(GAP)启动子的调控作用下,进行组成型分泌表达,在发酵液中获得了相对分子质量约45 kD的重组蛋白。摇瓶发酵结果表明,在发酵温度30℃、pH6.0、发酵时间72 h时重组蛋白表达占发酵上清液总蛋白68.4%。Western blotting显示,重组蛋白能够被相应抗体所识别。纯化后的重组hsRAGE具有与AGEs相互结合的活性,并能够显著地抑制AGEs诱导的人血管内皮细胞系ECV-304细胞NF-κB信号通路中NF-κB/p65基因的表达。     

乙型肝炎病毒包膜M蛋白在巴斯德毕赤酵母中的表达

转化了乙肝病毒 preS2 S基因的重组巴斯德毕赤酵母菌株经甘油培养基充分增殖 ,然后转移到甲醇培养基中进行诱导表达。破碎细胞并提取胞内蛋白 ,经ELISA和WesternBlot检测 ,证明有 4株GS1 1 5 /HIS MUT 表达了HBVM蛋白。     

Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris

We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2–10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF‐β proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

脂肪酶1在毕赤酵母中的高效表达

根据褶皱假丝酵母脂肪酶CRL中LIP1的成熟多肽序列 ,人工合成了由毕赤酵母 (Pichiapastoris)中偏爱密码子组成的lip1基因序列。该基因以N端融合的方式分别正确插入毕赤酵母诱导型表达载体pPICZαA与组成型表达载体pGAPZαA中。通过电激将线性化的上述两种重组质粒分别转化毕赤酵母SMD116 8H细胞 ,筛选获得两株分别具有诱导型表达和组成型表达生物活性LIP1能力的高产菌株。其中 ,组成型表达菌株CHT II换液后 4 8h上清液中含脂肪酶活力为 2 .0 0× 10 5u/L ,表达产物在pH 4～ 8与温度 30～ 5 0℃范围内具有较高的脂肪酶活性 ;高密度发酵条件下 ,发酵 72h ,上清液中含脂肪酶活力可达 1.395× 10 6u/L ,表明构建的重组菌株具有更大的工业化生产优势。