Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

Production and characterization of human extracellular superoxide dismutase in the methylotrophic yeast Pichia pastoris

Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H(2)O(2) oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.     

在毕赤酵母中利用口蹄疫病毒(FMDV)2A实现dTomato和串联MagaininⅡ基因的共表达

抗菌肽(antibacterial peptides)是生物体内经诱导产生的一种具有生物活性的多肽,是天然免疫的重要组成部分.为了提高抗菌肽的表达量以及很方便地检测抗菌肽的表达情况,本研究利用口蹄疫病毒(Foot-and-mouth disease virus,FMDV) 2A将荧光蛋白dTomato基因和3个串联的MagaininⅡ基因融合到一个开放阅读框中(open reading frame,ORE),融合的基因被置于pPIC9K载体醇氧化酶基因(AOX1)启动子的下游,构建分泌型重组酵母表达载体pPIC9K-dTomato -2A-3M,将线性化的重组酵母表达载体通过电击法转入毕赤酵母(Pichia pastoris )GS115中,经G418筛选和PCR鉴定得到阳性转化子,然后将其转至摇瓶,在30℃、0.5％甲醇的条件下进行诱导表达,连续诱导3d.SDS-PAGE电泳图谱显示,在31 kD(dTomato)和9.5kD(3M)处有蛋白条带出现,在荧光显微镜下观察酵母表达上清发出红色荧光,对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)抑菌试验结果表明,重组抗菌肽串联的MagaininⅡ具有明显的抑菌效果.结果表明,由2A连接的融合基因(荧光蛋白dTomato和串联的MagaininⅡ)在毕赤酵母中成功的进行了表达,FMDV 2A在其C端剪切多聚蛋白,得到dTomato和串联的MagaininⅡ两个独立而有活性的蛋白,为小分子抗菌肽的表达提供一个高效的检测方法.     

New strategy for expression of recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71

Gelatin is a well-known biopolymer, and it has a long history of use mainly as a gelling agent in the food industry. This paper reports a new method for producing recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71. Three independent expression cassettes encoding for specific length of gelatin, prolyl 4-hydroxylase (P4H, EC 1.14.11.2), α-subunit (αP4H), and protein-disulfide isomerase (PDI) were individually cloned in one expression vector, pPIC9K. The modified gelatin gene and two subunit genes of P4H were under the control of two different inducible promoters, namely, alcohol oxidase 1 promoter (PAOX1) and formaldehyde dehydrogenase 1 promoter (PFLD1), respectively. The results of sodium dodecylsulfate-polyacrylamide gel electrophoresis show that a recombinant gelatin was successfully expressed in P. pastoris KM71 by methanol induction. Liquid chromatography coupled with tandem mass spectrometry analysis indicates that the expressed gelatin was hydroxylated with approximately 66.7% of proline residues in the Y positions of Gly-X-Y triplets. The results of nuclear magnetic resonance spectroscopy of recombinant gelatin test show that the (1)H and (13)C spectra have many corresponding characteristic displacement peaks, and amino acids composition analysis shows that it contains hydroxyproline and its UV absorption is consistent with the characteristics of gelatin.     

以增加催化结构域的基因重构方法提高里氏木霉外切型葡聚糖酶Ⅱ的活性

分别通过增加CBHⅡ和EGⅣ催化结构域的方式对里氏木霉QM9414外切型葡聚糖酶Ⅱ进行基因重构。第一种重构方式是在CBHⅡ基因的上游增加EGⅣ的催化结构域基因，第二种重构方式是在CBHⅡ基因的下游增加其自身的催化结构域基因。通过PCR和基因重构手段获得重组质粒pPICZαA -cdE –cbh2和pPICZαA- cbh2-cdC，并在毕赤酵母GS115中表达，获得重组菌株P.pastoris PEC11和P.pastoris PCC16，在经改良的摇瓶培养条件下能表达具有较高CMCNa酶活性的融合蛋白，培养液的CMC活性分别达到3.87U/ml和7.66U/ml，其中P.pastoris PCC16表达的重构酶活性是毕赤酵母表达的单一CBHⅡ酶活性的2倍。表明采用增加结构域的方式可以有效提高纤维素酶的活性。     

经改造的猪IGF-I基因在巴斯德毕赤酵母中的分泌表达

采用酵母偏爱密码子合成五条编码猪IGF-I基因的引物，利用重叠PCR技术拼接获得长度为210bp的猪IGF-I成熟蛋白基因。将该基因插入分泌表达载体pPIC9K中，转化巴斯德毕赤酵母菌。经表型鉴定、PCR分析及G418筛选得到Muts型多拷贝整合菌，以1.5%甲醇诱导培养后，经SDS-PAGE检测表达产物，在7.5 kDa处出现一特异蛋白条带，目的蛋白表达量达410mg/L。Dot blot检测表明，重组蛋白可与抗IGF-I多克隆抗体反应。     

Cloning and expression of the XPR2 gene from Yarrowia lipolytica in Pichia pastoris

Yarrowia lipolytica is a dimorphic yeast able to secrete different types of proteases depending on the pH of the environment. At neutral pH, the production of an extracellular alkaline protease (AEP) is induced. This protease could be useful in the leather, detergent, or food industries. The XPR2 gene, coding for AEP, was extracted from the pINA154 vector and cloned into the pHIL-D2 vector to obtain a new protease-producing recombinant Pichia pastoris strain. The gene was efficiently integrated in the P. pastoris genome and expressed from the AOX1 promoter actively induced by methanol. Finally, the protease was successfully secreted by P. pastoris GS115.     

Expression and purification of glycosylated bovine beta-casein (L70S/P71S) in Pichia pastoris

Post-translational glycosylation of bovine beta-casein (L70S/P71S) that results in Asn(68)-linked glycan on the protein was obtained in up to 30% of total beta-casein expressed in the methylotrophic yeast Pichia pastoris. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine beta-casein, indicating pH-dependent glycosylation. Glycosylated bovine beta-casein (L70S/P71S) expressed in P. pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A--Sepharose affinity column, and Mono Q anion-exchange FPLC. The purified glycosylated bovine beta-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine beta-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine beta-casein was normally phosphorylated to the same degree as native bovine beta-casein.     

里氏木霉木聚糖酶基因Ⅱ在毕赤酵母中的分泌表达及其酶学性质研究

利用RT-PCR技术从高产木聚糖酶菌株T. reesei Rut C-30中成功扩增出其主要木聚糖酶基因XynⅡ,经序列分析证实,在该菌株诱变选育过程中其成熟肽序列有两处碱基发生突变;将不带原基因信号肽的XynⅡ克隆到毕赤酵母高效表达载体pPICZαA上,线性化后经电击转化到毕赤酵母中,经Zeocin及PCR筛选后得到的转化子用1%甲醇诱导。SDS-PAGE证实,重组子实现了分泌表达,且发酵上清中几无杂蛋白;对该重组酶酶学性质分析表明,该酶最适反应温度为60℃,最适反应pH值为6.0,该酶热稳定性较好,在50℃下保温30 min仍能保留其95%以上活性。     

锰超氧化物歧化酶（Mn-SOD）基因的分子改造及在毕赤酵母中的表达

根据毕赤酵母（Pichia pastoris）的密码子偏好性,以不改变氨基酸序列为原则,对源于蜡样芽孢杆菌M22（Bacillus cerues M22）的Mn-SOD基因进行分子改造,设计、合成新的基因序列Mn-SOD-2。构建酵母表达载体pPICZαA/Mn-SOD-2,并整合至毕赤酵母GS115染色体。结果表明,所构建的酵母工程菌株YM103,经0.5%甲醇诱导表达后,Native-PAGE检测证实有清晰活性条带;SDS-PAGE检测证实重组蛋白的分子量为24kD,与预期大小一致。酶活分析表明,外源蛋白的活性较改造前增加了2.2倍,且表达稳定性良好。     

抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

抗菌肽模式肽PGYa的分子设计、克隆及在毕赤酵母中的分泌表达

在α-螺旋抗菌肽序列比较和两亲性分析的基础上,提取序列模板,计算机辅助（螺旋轮法）设计出新型抗菌肽模式肽PGYa(Peptide以Gly开头,以Tyr-NH2结尾),然后选用毕赤酵母偏爱密码子,设计合成了PGYa基因（rPCR法）。所合成的基因全长为94bp,并在其N端引入kex2裂解位点,以保证表达抗菌肽具有天然N端。基因克隆入pPICZα-A质粒,构建分泌型酵母表达载体pPICZα-A-PGYa。在AOX1 (醇氧化酶)启动子调控下,PGYa蛋白获得分泌表达,其表达量达到132 mg/L。初步抑菌（E.coli DH5α）活性显示：PGYa有较好的抗菌活性。     

Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastoris

It was shown previously that a bacterially expressed mungbean defensin VrCRP exhibited both antifungal and insecticidal activities. To isolate this protein in a large quantity for its characterization, the defensin cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified. The recombinant VrD1 was shown to inhibit the growth of fungi such as Fusarium oxysporum, Pyricularia oryza, Rhizoctonia solani, and Trichophyton rubrum and development of bruchid larva. The protein also inhibits in vitro protein synthesis. These biological activities are similar to that of the bacterially expressed defensin. Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of VrD1 using the mutagenesis approach.     

Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme

Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZalphaC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.     

Effect of glycosylation modification (N-Q-(108)I --> N-Q-(108)T) on the freezing stability of recombinant chicken Cystatin overexpressed in Pichia pastoris X-33

The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn(106)-Ile(108)-->Asn(106)-Thr(108)) were cloned into the pGAPZ alpha C expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host. The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated. A large quantity of recombinant chicken cystatin and the Asn(106)-glycosylated cystatins were expressed and secreted into broth using alpha-factor preprosequence. The K(i) of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM). They acted as a competitive inhibition reaction against papain. According to the K(i), the inhibition ability of Asn(106)-glycosylated mutant cystatin (K(i) = 9.5 nM) was weaker than that of the wild-type one. However, N-glycosylation at Asn(106) substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P. pastoris.     

Engineering the expression and biochemical characteristics of recombinant Candida rugosa LIP2 lipase by removing the additional N-terminal peptide and regional codon optimization

Candida rugosa lipase (CRL), an important industrial enzyme, has been established, containing several different isoforms which were encoded by the high-identity lip gene family (lip1 to lip7). In this study, we compared the expression and biochemical characterization with three different engineered lip2 constructions in the yeast Pichia pastoris. Our results showed that lip2 (lip2) has an overall improvement of 50% higher production yield (1.446 U/mL) relative to that of nflip2 (0.964 U/mL) at 7 days of cultivation time. Codon-optimized lip2 (colip2) has a 2.3-fold higher production yield (2.182 U/mL) compared to that of lip2 (noncodon-optimized; 1.446 U/mL) and nflip2 (0.964 U/mL), with a cultivation time of 5 days. This finding demonstrated that the removal of the N-terminus and the regional codon optimization of the lip2 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP2 in the P. pastoris system. The distinct biochemical properties of our purified recombinant nfLIP2 and LIP2 suggested that they are potentially useful for various industrial applications.     

犬白细胞介素2在毕赤酵母中的诱导表达及生物活性的测定

以ConA刺激的犬外周血淋巴细胞总RNA为模板，通过RT-PCR方法扩增出犬IL-2成熟蛋白基因，将目的片段连接到pMD18-T载体，测序结果显示，扩增片段与GenBank上发表的序列一致。然后将目的片段连接到酵母表达载体pPICZa-A上，得到重组酵母犬IL-2表达载体pPICZaA-CaIL-2，经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。PCR方法筛选重组酵母菌，甲醇诱导表达，SDS-PAGE结果显示表达上清中有大小约20kDa的目的条带，比实际分子量略大，推测蛋白可能发生糖基化。MTT法测定生物学活性结果表明，重组犬IL-2能够极显著促进犬外周血淋巴细胞增殖。证明酵母表达的犬重组IL-2具有良好的生物学活性。     

Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.     

Expression and characterization of sweet potato invertase in Pichia pastoris

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.