桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。

Heterologous expression of a dipeptidase from Inonotus obliquus in Pichia pastoris

The heterologous expression of a dipeptidase (idp)-encoding gene from medicinal basidiomycete Inonotus obliquus using Pichia pastoris expression system is described. The gene contains an encoding region of 1 814bp nucleotides, which contains six introns and encodes 465 amino acids (aa). Bioinformatic analysis indicates that the predicted amino sequence does not contain signal peptide but harbors a transmembrane region (TR) located between 55-77aa. The coding sequences with (idp) and without TR (didp) were amplified and respectively cloned into the secreting expression vector pPICZαA. The constructed plasmids were transformed into P. pastoris strain X-33 which was induced by 1% (V/V) methanol for iDP expression. SDS-PAGE and Western-blot analysis were used to detect expressed proteins. The gene idp either harboring TR region or not could be successfully expressed in Pichia pastoris. However, the expressed iDP encoded by whole idp cannot be secreted from yeast cells but presented in the sediments of cell debris with no catalytic activity. In contrast, the DP with the removal of TR (diDP) could be secreted from P. pastoris with the capacity to hydrolyze dipeptides. Expressed diDP was purified by Ni-NTA sepharose column chromatography with the protein concentration reaching to 0.12mg/mL. Its specific activity was 433U/mg when detected under the condition of pH 7.3 and 50°C for 2h using Gly-Gly as substrate, which also displayed high activity to hydrolyze dipeptides Ile-Leu, Trp-Trp and Phe-Phe.