人源Ⅲ型胶原蛋白在毕赤酵母中的多拷贝重组表达、鉴定及抗氧化活性分析

Recombinant Expression,Structural Identification,and Antioxidant Activity Analysis of Human Type III Collagen in Pichia pastoris

胶原蛋白在人体内有重要作用,并且在食品、保健品、医疗等方面有广泛应用。该研究针对毕赤酵母的密码子偏好性对人源Ⅲ型胶原蛋白基因进行了密码子优化,在此基础上构建了人源Ⅲ型胶原蛋白单串联、二串联和二串联四拷贝表达载体pPIC9K-COL3-S、pPIC9K-COL3-2和pPIC9K-COL3-4,转化毕赤酵母GS115实现了人源Ⅲ型胶原蛋白的整合表达,获得了胶原蛋白单串联、胶原蛋白二串联和胶原蛋白二串联四拷贝的毕赤酵母工程菌株。对pPIC9K-COL3-S、pPIC9K-COL3-2重组菌株进行了摇瓶模拟高密度发酵,甲醇诱导浓度为0.5%,经SDS-PAGE和Western Blot检测,重组菌株成功表达了重组胶原蛋白,其中单串联蛋白表观分子量约为26.7 ku,双串联蛋白表观分子量约为52.3 ku。四拷贝重组菌株在0.5%甲醇诱导下的高密度摇瓶发酵产量最高,在最佳诱导时间为72 h时,蛋白产量达到约0.45 g/L。通过镍柱纯化后获得高纯度重组蛋白,抗氧化活性实验表明,重组胶原蛋白DPPH自由基清除率达到51.49%、ABTS自由基清除率达到41.24%,证明具有抗氧化活性,为其在食品,保健品和医疗领域的应用提供理论依据。

Collagen plays a vital role in the human body and is widely used in food,health products,and medical treatment.Codon optimization of the human type III collagen gene was carried out according to the codon usage bias of Pichia pastoris.The single-tandem,two-tandem,and four-copy two-tandem expression vectors pPIC9K-COL3-S,pPIC9K-COL3-2,and pPIC9K-COL3-4,respectively,were constructed and transformed into P.pastoris GS115 to achieve integrated expression of human type III collagen,thereby obtaining engineered strains of P.pastoris containing single-tandem collagen,two-tandem collagen,and four-copy two-tandem collagen.The pPIC9K-COL3-S and pPIC9K-COL3-2 recombinant strains were mixed and shaken with a 0.5%induction concentration of methanol to stimulate high-density fermentation.SDS-PAGE and western blot analysis demonstrated that the recombinant strains successfully expressed recombinant collagen,where the apparent molecular weight of the single-tandem protein was approximately 26.7 ku,and that of the two-tandem protein was approximately 52.3 ku.The yield of the high-density shake flask fermentation of the four-copy recombinant strain induced by 0.5%methanol was the highest,with an optimal induction time of 72 h and protein yield reaching approximately 0.45 g/L.A high-purity recombinant protein of this strain was obtained after purification using a nickel column.Antioxidant activity experiments showed that the DPPH free-radical scavenging rate of the recombinant collagen reached 51.49%,whereas the ABTS free-radical scavenging rate reached 41.24%,thus proving its antioxidant activity.This provides a theoretical basis for its application in the fields of food,health products,and medicine.