Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry

The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme beta-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZ/V5-His, which was used for the constitutive expression in stably transformed Sf21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by sequential chromatography on Ni-NTA-agarose and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the structure (GlcNAc)(2)Fuc(Man)(3).     

SARS冠状病毒S蛋白结构预测及表达初探

利用生物信息学的工具和方法,对SARS病毒基因组中预测的S蛋白（spike protein）的编码序列进行了分析,预测了其结构特征和可能的功能域。选择其中最有可能参与受体识别及产生主要免疫原性的区域(S401～659)作为待表达区域。将通过PCR获得的此段S蛋白片段的编码序列克隆至大肠杆菌载体pET28a(+)和酵母表达载体pPIC9K中,分别构建了pET28a(+)-S和pPIC9K-S表达质粒,转化至大肠杆菌菌株BL21(DE3)-star和毕赤酵母中表达。产物的蛋白电泳和蛋白印记结果表明,S蛋白片段获得成功表达。采用镍离子螯合层析法纯化变性的包涵体样品,纯度达90%以上。     

Expression,purification, and characterization of recombinant human neurturin secreted from the yeast Pichia pastoris

Neurturin (NTN), a potent neurotrophic factor acting specifically on dopaminergic neurons, is comprised of 102 amino acids as a mature protein. We artificially synthesized a gene for mature human NTN (hNTN) using codons preferred by the yeast Pichia pastoris. This synthesized gene, fused in frame with sequences encoding the alpha-factor signal peptide gene from Saccharomyces cerevisiae was cloned into P. pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-hNTN was then transformed into the yeast and stable multicopy recombinant P. pastoris strains were selected by G418 resistance. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hNTN, a 16kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using CM-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant hNTN was confirmed by the ability of the protein to stimulate growth of nerve fibers from the dorsal root ganglia of chick embryos in vitro.     

昆虫神经毒素LqhIT2的表达、抗血清制备及活性分析

根据毕赤酵母密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素LqhIT2基因,并分别克隆至大肠杆菌融合表达载体pPET30-a( )和毕赤酵母分泌表达载体pPIC9K.在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化后,用于免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度超过1:128 000.利用制备的抗血清,采用斑点杂交,筛选得到了较高水平分泌表达重组LqhIT2的酵母转化子,摇瓶条件下,毒素表达量约9 mg/L.大肠杆菌表达产物没有生物活性,酵母表达产物经注射蝗虫表现出杀虫活性.     

猪囊尾蚴疫苗候选基因TSO18在酵母中的高效表达

将猪带绦虫六钩蚴TSO18基因亚克隆至毕赤酵母分泌性表达载体pPIC9K,构建重组表达载体pPIC9K_TSO18,电转化毕赤酵母菌GS115,使重组表达载体与酵母染色体发生同源整合。采用G418抗性梯度法筛选得到多拷贝重组菌株,用甲醇进行诱导表达,并对表达产物进行SDS_PAGE和Western blot分析、脱糖基化分析、分子筛纯化和小鼠免疫接种等表明,目的蛋白得到了高效表达并进行了适度的糖基化,易于纯化且具有免疫活性。在5L发酵罐中目的蛋白表达量达到2.54mg/mL,为制备基因工程疫苗打下了坚实的基础。     

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.     

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.     

High-level expression and purification of recombinant human catalase in Pichia pastoris

Catalase is one of the antioxidant enzymes and is involved in many pathophysiologic processes and human diseases. This study focused on high-level expression and purification of recombinant catalase in Pichia pastoris. The cDNA encoding catalase was cloned by RT-PCR from Fetal liver of Homo sapiens. After PCR and construction of expression vector pPIC9K-CAT, human catalase was expressed highly in P. pastoris yeast SMD1168 and secreted into the culture medium. The secreted catalase was purified to a purity of 95% by ammonium sulfate fractionation, anionic exchange-chromatography, and Macro-prep Ceramic Hydroxyapatite with a overall yield of 60%. This study provides a new method for large-scale expression and purification of recombinant protein catalase.     

Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris

Ancrod is known as a thrombin-like enzyme from the venom of Calloselasma rhodostoma. The cDNA encoding ancrod was synthesized with a yeast bias codon and inserted into the eukaryotic expression vector pPIC9 and was subsequently expressed in the yeast Pichia pastoris. Recombinant ancrod was produced in 5-L bioreactor using a sorbitol-methanol feeding strategy and recovered from the fermentation broth by hydrophobic, affinity, and ion exchange chromatography. SDS-PAGE analysis revealed that ancrod was heterogeneously glycosylated and running at the expected molecular weight of 43-48 kDa which decreased to about 29 kDa after deglycosylation with N-glycosidase F. The fibrinogenolytic and zymographic activity of the recombinant ancrod were determined and were found to be similar to that of the native protein.     

hCGβ-CTP37多聚体在巴斯德毕赤酵母中的表达及其表达产物的结构预测

hCGβ-CTP37(简称CTP)肽段是hCG分子的特有部分,存在hCG的特异表位。为了解CTP利用毕赤酵母进行表达的情况及探索其作为研制调节生育和抗肿瘤疫苗的可行性,本研究中我们将2、3、4个CTP以头尾串联的方式重组成多聚体基因,然后克隆入载体pPIC9K得到表达质粒pPIC9K-Cα-(CTP)n(n=2,3,4),电转染入酵母细胞进行表达。在培养基上清中我们检测到了目的基因表达产物,Western blot结果显示它们的分子量分别约为：20KDa、30KDa、40KDa,且能与抗hCG多克隆抗体结合。另外,我们还利用ANTHEPROT 4．3蛋白序列分析软件包对表达产物CTP四聚体的结构进行了预测分析,结果表明CTP四聚体的抗原性明显强于hCGβ-CTP37单聚体,CTP四聚体与hCGβ二级结构较为相似,但CTP四聚体的抗原专一性要优于hCGβ。CTP多聚体在毕赤酵母系统中的成功表达及对表达产物抗原性的初步分析为今后利用CTP研制调节生育和抗肿瘤疫苗奠定了基础。     

重组人kallistatin蛋白在毕赤酵母中的高效表达及生物活性分析

为了研究kallistatin(Kal)的生物活性,本实验构建了可分泌表达Kal的毕赤酵母菌株。首先通过PCR方法从pAAV-Kal中扩增出KalcDNA,并克隆至酵母表达载体pPIC9,得到甲醇酵母分泌型表达载体pPIC9-Kal,然后将载体线性化并电击转化毕赤酵母GS115(his4),通过MD平板筛选出阳性表达菌株。阳性表达菌株在BMMY培养基(pH7.0)中29℃培养,经2%甲醇诱导表达96h,摇瓶表达量可达14mg/L。表达上清经PhenylSuperose、Heparin SepharoseFF分离纯化,目的蛋白纯度达到98%,分子量为58kDa。生物活性实验显示,所得到的Kal蛋白具有较好的抗氧化活性,过氧化物酶活性达到(163±4)U/(mg·min),可有效降低H2O2对LX-2细胞的氧化损伤。另外,重组产生的Kal还能抑制HUVEC细胞的增殖。本研究首次成功地利用毕赤酵母表达系统分泌表达了有生物活性的Kal,为继续开展其抗肿瘤活性奠定了基础。     

来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达

柠檬酸杆菌(Citrobacterbraakii)来源的植酸酶是目前报道的比活最高的植酸酶。按照毕赤酵母(Pichiapastoris)对密码子的选择偏向性,对来源于柠檬酸杆菌的高比活植酸酶基因AppA进行了密码子优化改造。改造后的基因AppA(m)按正确的阅读框架融合到毕赤酵母表达载体pPIC9的α-因子信号肽编码序列3′端,通过电击转化得到重组转化子。通过PCR验证,AppA(m)已整合在酵母染色体上。SDS-PAGE分析和表达产物的研究表明,植酸酶得到了高效分泌表达,在5L发酵罐中植酸酶蛋白表达量达到3·2mg/mL发酵液,发酵效价达到每毫升发酵液1·4×107IU以上,高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

丙型肝炎病毒核心蛋白基因在毕赤酵母中克隆与分析

目的:克隆丙型肝炎病毒核心蛋白基因及其上游DNA序列,为此基因的表达研究作准备。方法:用反转录和PCR方法从HCV的总RNA中扩增得到核心蛋白基因及其上游DNA序列,连接到pMD18-T载体上,用限制性内切酶切下目的基因,插入到巴斯德毕赤酵母表达载体pPIC9K中,构建成重组质粒,测序证明正确后,再将目的基因在毕赤酵母中进行克隆,鉴定。结果:重组质粒转化毕赤酵母后,经PCR鉴定,证明形成了目的基因的克隆。结论:应用毕赤酵母作为受体菌,pPIC9K为载体,成功克隆了HCV核心蛋白基因。     

Expression and Antigenic Characterization of the Epitope-G1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

The epitope-G1 gene of Bovine ephemeral fever virus(BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1.Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115.The recombinant P.pastoris strains were selected by a G418 transformation screen and confirmed by PCR.After being induced with methanol,an expressed protein with 26 kDa molecular weight was obtained,which was much bigger than the predicted size(15.54 kDa).Deglycosylation analysis indicated the recombinant G1 was glycosylated.Western blot and ELISA tests,as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity.The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.     

High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris

The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.     

乙酰氨基半乳糖转移酶14(GALNT14)在毕赤酵母中的表达纯化及活性鉴定

将GALNT14全长编码区克隆到分泌型酵母表达载体pPIC9K中,构建重组载体pPIC9K-T14,经电转至毕赤酵母GS115中表达,使用G418筛选高表达重组菌,并对诱导条件进行优化,表达产物使用SDS-PAGE分析、Western blot鉴定、Sephadex G-100纯化、最后经HPLC检测活性。结果显示,在提供更好的供氧量条件下,用0.75％甲醇诱导可提高目的蛋白的表达量而降低杂蛋白的表达。培养上清液经SDS-PAGE检测显示目的蛋白分子量约64 kDa;Western blot结果显示在相应分子量处有一条特异性条带;利用Sephadex G-100成功分离纯化了目的蛋白;活性测定结果显示所获得的目的蛋白具有催化活性,为深入研究GALNT14的结构与功能奠定了基础。     

High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris

Apolipoprotein AI (ApoAI) is an important apolipoprotein in plasma and is known to have various physiological functions suitable for pharmaceutical applications. Human blood has been the only source of this protein for research and large-scale applications. To obtain large amounts of ApoAI a Pichia pastoris expression system was first used to obtain a high level of expression of secreted, recombinant protein. The human gene encoding ApoAI was inserted into the secretion vector pPIC9K and used to transform P. pastoris GS115. AP16, a high expression transformant with high G418 resistance, was obtained. After induction with methanol, the expression level of rhApoAI (recombinant human ApoAI) was 160 mg/L in a 14L fermentor. RhApoAI was purified by cold acetone precipitation followed by Q-Sepharose Fast Flow ion exchange column chromatography with 60% recovery. The N-terminal amino acid sequence and molecular weight (mass spec.) of rhApoAI are identical to native human ApoAI. Purified rhApoAI has specific binding activity with liver cells SMC7721 and binding can be inhibited by native human ApoAI.     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G1抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G1,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

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.     

Intein介导的重组昆虫毒素BmK IT在大肠杆菌中的可溶性表达、纯化及活性分析

为获得重组蝎昆虫毒素BmKIT,通过PCR方法在BmKIT基因的3′端融合了编码6个组氨酸残基的核苷酸序列,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB Intein基因下游的多克隆位点(MCS)。将获得的表达质粒转化大肠杆菌BL21(DE3)中,用IPTG诱导融合蛋白表达。用Ni-NTA亲和层析柱从菌体裂解液中纯化了CBD-Intein-BmK IThis6融合蛋白,并在柱上诱导Intein自剪切,成功去除融合子CBD-Intein。通过Superdex75凝胶过滤层析获得了纯度达95%以上的BmK IThis6蛋白,该蛋白不仅具有正确的二级结构而且有生物活性。