嗜甲醇酵母菌Pichia pastoris醱酵生產重組T4 endonuclease V之研究

Abstract

T4 endonuclease V (T4N5)是T4 bacteriophage中的DNA修復酵素，為大小16 kDa的蛋白質，可專一性辨認因紫外線造成DNA損傷而形成的環丁烷嘧啶二聚體(cyclobutane pyrimidine dimer, CPD)並切除，再由後續DNA修復機制進行修補。過去研究利用大腸桿菌生產重組T4N5，結合微脂體(Liposome)技術製成乳劑，針對缺乏核苷酸切除修復(nucleotide excision repair)能力的患者進行臨床實驗，發現可減少病灶生成，顯示T4N5移除紫外線造成的DNA損傷功能對人體亦有所幫助。為使此技術能廣泛應用於防曬乳及保養品中，提升產品附加價值，須尋求低成本大量生產重組T4N5的方法。現行的重組T4N5多以大腸桿菌進行生產，然而欲取得目標蛋白質須經破菌及多道純化手續，且有內毒素的疑慮。嗜甲醇酵母菌Pichia pastoris為單細胞真菌表達系統，可進行高密度培養，生產基因重組蛋白質成本相較低廉，同時沒有內毒素疑慮並可利用外泌訊息胜肽將目標蛋白質分泌至培養基中以方便純化。本研究利用嗜甲醇酵母菌P. pastoris KM71H作為重組T4N5表達系統，以搖瓶和醱酵槽進行培養，添加甲醇作為碳源並誘導異源蛋白質表現。搖瓶培養可成功外泌表現出序列正確且具活性的T4N5，活性可達0.47±0.05 U/µL。但以醱酵槽進行高細胞密度培養時，發現以2.6 mL/L/h的速率添加甲醇可能會對菌體造成壓力使部分菌體死亡並裂解釋放胞內蛋白酶，但減低甲醇添加量又不利於菌體生產異源蛋白質，產量皆不理想，須以其他方式減少菌體死亡裂解及抑制蛋白酶作用。本研究發現，同樣以約2.6 mL/L/h的速率添加甲醇，將誘導溫度由30oC降低至 23oC，可減低培養基中的蛋白酶含量，並增進甲醇異化代謝路徑，大幅提升目標蛋白質表現量。

T4 endonuclease V (T4N5) is a DNA repair enzyme isolated from T4 bacteriophage. It can specifically recognize and remove cyclobutane pyrimidine dimer which is resulted from the UV-induced DNA damage and might cause skin cancer. In previous reports, in conjunction of recombinant T4N5 expressed by Escherichia coli and liposome, T4N5 liposome lotion effectively penetrated into skin in vivo and clinical trials also indicated its potential for UV-induced DNA damage treatment. In order to extensive application of T4N5 in skin care products such as sunscreen, it’s important to develop a method for large-scale production recombinant T4N5 with low expenses. The major recombinant T4N5 are currently produced by E. coli. However, the procedures include cell lysis and protein purification, which may increase the production cost. Moreover, it is at risk due to endotoxins. Pichia pastoris is a well-defined expression system which can be cultivated at high cell density at low cost. In this study, P. pastoris KM71H was used as a heterologous expression system, and methanol was added to induce recombinant T4N5 expression. In flask, we have successfully expressed extracellular T4N5 with correct conformation and function. The T4N5 activity in supernatant of flask culture reached up to 0.47±0.05 U/µL. When using 5 L fermenter, methanol fed at 2.6 mL//L/h induced cell lysis and release of cellular proteases, while reducing methanol feeding rate led to low yield of target protein yield. Therefore, it’s necessary to modify other induction conditions. In this study, we found that reduction of the induction temperature from 30oC to 23oC improved recombinant protein yield remarkably at the same methanol feeding rate. The amount of proteases in the culture decreased, and methanol dissimilation is improved.