小麦过氧化物酶（WP1）在毕赤酵母中的表达、纯化及活性鉴定
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摘要
面筋含量是影响面粉加工品质的决定性因素。面筋的三维蛋白网络骨架是由小麦高分子量麦谷蛋白(HMW-GS)、低分子量麦谷蛋白(LMW-GS)和醇溶蛋白(Gliadins)交联形成。2001年Tilley等人提出了面筋蛋白网络形成的酪氨酸交联理论,并证明面粉中存在可催化面筋蛋白酪氨酸交联的酶组分。本研究组已从小麦种子中纯化到一种具有酪氨酸交联活性的过氧化物酶WP1,并克隆到该酶的cDNA。本研究在上述研究的基础上将小麦WP1基因重组到毕赤酵母(P. pastoris)基因组中,并对重组蛋白进行了初步纯化和活性鉴定。
毕赤酵母(P. pastoris)表达系统是目前较完善的高效异源真核蛋白表达系统,具有高分泌表达、高稳定整合、易于高密度发酵和产物的下游纯化等优点。本研究通过PCR在WP1基因3’-末端添加His-tag编码序列;克隆鉴定后的WP1-His编码序列经双酶切、连接转化等步骤重组到毕赤酵母表达载体pPIC9K中;再将限制性内切酶BglⅡ线性化的载体通过电转化导入毕赤酵母GS115,在组氨酸缺陷的MD平板进行初步筛选鉴定,再用愈创木酚测活法筛选高表达菌株,最终从200个单克隆中筛选出5株有明显过氧化物活性重组克隆。扩大表达后用Ni-NTA亲和柱进行分离纯化,用愈创木酚法测活结果表明,分离到的WP1具有过氧化物酶活性,纯化的洗脱液用SDS-PAGE检测纯化到和预期蛋白条带大小相同的条带,进一步确定了本研究确定了WP1蛋白在毕赤酵母中的高校表达。
本研究成功地在毕赤酵母中表达了具有活性的小麦WP1,确立了较完善的毕赤酵母表达体系。本研究为WP1与小麦加工品质关系的阐明,以及WP1在食品工业中的潜在应用奠定了基础。
Gluten content determines the process quantity of the flour. HMW-GS, LMW-GS and Gliadins cross link together to make up gluten’s 3-dimension protein network skeleton. Since 2001, Tilley etc. first fingered out the theory of tyrosine cross-linking in the Gluten. This result was supported by lots of research results. The researches find out it is several peroxdiases or enzymes which have oxidase characteristic catalyzed form of dityrosine. We have purified one kind of peroxidase (WP1) which has the activity to catalyze tyrosine crosslinking, and it’s cDNA was cloned. In this study, I tried to express active WP1 protein in Pichia pastoris, the purpose of this research is try to find a potential safe and cheap food additive, and it has very huge practical value for the food industry.
P. pastoris has many of the advantages of higher eukaryotic expression systems such as high secretion expression, stable integration in the P. pastoris genomic gene, easy high density fermentation and production downstream purification and so on. My research first is to amplify WP1 gene form the clone vector, which been save in our lab. Then through digest, ligation and transformation procedure clone WP1 gene to the P. pastoris expression vector pPIC9K. The next step was linear the plasmid then use high efficiency electroporation to transfer the gene into the P. pastoris . Through homologous recombination linear pPIC9K with WP1 gene was integrated into Pichia genomic gene. Positive recombinant expression bacteria were select by MD plate. Through comparing activity among the positive clones on the MD plate to select high expression recombinant cell by Guaiacol method. WP1 was purified by His-tag Ni-NTA chromatography column and the purification product has peroxidase activity. WP1 purification product was checked by SDS-PAGE, the size of brand is the as expected, this result further affirmed WP1 protein was high yield in P. pastoris
This research successful expressed active WP1 protein in Pichia pastoris. Lay the foundation for the food industry to fond safe and cheap additives.
毕赤酵母(P. pastoris)表达系统是目前较完善的高效异源真核蛋白表达系统,具有高分泌表达、高稳定整合、易于高密度发酵和产物的下游纯化等优点。本研究通过PCR在WP1基因3’-末端添加His-tag编码序列;克隆鉴定后的WP1-His编码序列经双酶切、连接转化等步骤重组到毕赤酵母表达载体pPIC9K中;再将限制性内切酶BglⅡ线性化的载体通过电转化导入毕赤酵母GS115,在组氨酸缺陷的MD平板进行初步筛选鉴定,再用愈创木酚测活法筛选高表达菌株,最终从200个单克隆中筛选出5株有明显过氧化物活性重组克隆。扩大表达后用Ni-NTA亲和柱进行分离纯化,用愈创木酚法测活结果表明,分离到的WP1具有过氧化物酶活性,纯化的洗脱液用SDS-PAGE检测纯化到和预期蛋白条带大小相同的条带,进一步确定了本研究确定了WP1蛋白在毕赤酵母中的高校表达。
本研究成功地在毕赤酵母中表达了具有活性的小麦WP1,确立了较完善的毕赤酵母表达体系。本研究为WP1与小麦加工品质关系的阐明,以及WP1在食品工业中的潜在应用奠定了基础。
Gluten content determines the process quantity of the flour. HMW-GS, LMW-GS and Gliadins cross link together to make up gluten’s 3-dimension protein network skeleton. Since 2001, Tilley etc. first fingered out the theory of tyrosine cross-linking in the Gluten. This result was supported by lots of research results. The researches find out it is several peroxdiases or enzymes which have oxidase characteristic catalyzed form of dityrosine. We have purified one kind of peroxidase (WP1) which has the activity to catalyze tyrosine crosslinking, and it’s cDNA was cloned. In this study, I tried to express active WP1 protein in Pichia pastoris, the purpose of this research is try to find a potential safe and cheap food additive, and it has very huge practical value for the food industry.
P. pastoris has many of the advantages of higher eukaryotic expression systems such as high secretion expression, stable integration in the P. pastoris genomic gene, easy high density fermentation and production downstream purification and so on. My research first is to amplify WP1 gene form the clone vector, which been save in our lab. Then through digest, ligation and transformation procedure clone WP1 gene to the P. pastoris expression vector pPIC9K. The next step was linear the plasmid then use high efficiency electroporation to transfer the gene into the P. pastoris . Through homologous recombination linear pPIC9K with WP1 gene was integrated into Pichia genomic gene. Positive recombinant expression bacteria were select by MD plate. Through comparing activity among the positive clones on the MD plate to select high expression recombinant cell by Guaiacol method. WP1 was purified by His-tag Ni-NTA chromatography column and the purification product has peroxidase activity. WP1 purification product was checked by SDS-PAGE, the size of brand is the as expected, this result further affirmed WP1 protein was high yield in P. pastoris
This research successful expressed active WP1 protein in Pichia pastoris. Lay the foundation for the food industry to fond safe and cheap additives.
引文
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