牛透明质酸酶PH-20核心区在毕赤酵母中的高效表达

High-level Expression of Core-domain Coding Sequence of Hyaluronidase PH-20 from Bull Testis in Pichia Pastoris

目的利用毕赤酵母表达牛源透明质酸酶核心区,以实现透明质酸酶的重组制备。方法将去除信号肽和锚定区域的牛源透明质酸酶PH-20成熟肽编码序列按照毕赤酵母密码子偏爱性进行优化,然后连入pPICZaA表达载体并转入毕赤酵母SMD1168H菌株。0.5%(v/v)甲醇诱导表达72 h后采用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)法测定培养液上清中透明质酸酶活性。结果包含PH-20核心序列(1332 bp)的重组酵母经甲醇诱导表达72 h,培养液上清中透明质酸酶活性达135.2 U/mL。结论实现了牛源透明质酸酶基因在毕赤酵母中的高效表达。

Objective To express the core domain coding sequence of hyaluronidase PH-20 from bull testis in Pichia pastoris, and to achieve the recombinant expression of PH-20 protein. Methods The core domain coding sequence of PH-20 without signal and anchor sequences was optimized according to the codon usage preference of P. pastoris, ligated with plasmid pPICZaA and transformed into P. pastoris SMD 1168H. After induced by 0.5 % （v/v） methanol for 72 h, the hyaluronidase activity in the supernatant was determined using 3, 5-dinitrosalicylic acid （DNS） method. Results Recombinant yeast strain which contained the core domain coding sequence of PH-20 was induced by 0.5 % （v/v） methanol for 72 h, and the hyaluronidase activity in the supernatant was up to 135.2 U/mL. Conclusion PH- 20 from bull testis can be high-levelly expressed in P. pastoris.