海藻糖合酶基因在毕赤酵母中的表达

Expression of Trehalose Synthase in Pichia pastoris

摘要:

首次构建来源于恶臭假单胞菌海藻糖合酶基因（AE015451.1）的真核表达载体，探索在毕赤酵母中的表达。PCR扩增目的基因，经EcoRⅠ/XbaⅠ双酶切后连接至同样双酶切的pPICZaA中，经PCR检测和序列测定准确后，通过电击法将重组质粒转入毕赤酵母GS115中，利用含Zeocin的YPD平板筛选到阳性转化子，提取阳性转化子基因组并利用特异性引物PCR得到一条与目的基因大小相同的条带，说明目的基因转入成功，诱导重组蛋白表达并用SDS-PAGE检测可见一条约76 ku与目的蛋白大小预测相符合的蛋白条带，最后HPLC检测重组蛋白可将麦芽糖特异转化为海藻糖，说明外源表达的海藻糖合酶具有一定酶活。通过PCR、SDS-PAGE和HPLC结果说明成功构建重组pPICZaA-TreS质粒并整合到巴斯德毕赤酵母的基因组上，且海藻糖合酶基因得到预期的表达并具有活性，为下一步研究奠定基础。

Abstract:

The expression vector was constructed from trehalose synthase (TreS) gene（AE015451.1）of Pseudomonas putida to express in Pichia pastoris. TreS gene was amplified by PCR and double digested by EcoRⅠ/XbaⅠ, then linked to the pPICZaA which digested with same enzymes. The gene was identified, sequenced and transformed into pichia pastoris GS115 by electroporation. Positive colonies harboring target genes were screened out by the YPD medium with Zeocin. The genome of positive colonies were extracted and a same size band with target gene was abtained by using PCR which illustrated target gene had successfully transferred to Pichia pastoris. SDS-PAGE and HPLC results showed that the recombinant enzyme had a molecular of 76 ku band which was consistent with the predicted molecular mass, and it had the ability to catalyze the conversion of maltose into trehalose. The recombinant pPICZaA-TreS plasmid successfully constructed and integrated into Pichia pastoris genome and expressed as expected.