重组人α1-微球蛋白的毕赤酵母发酵与纯化工艺优化  

Optimization of the Fermentation and Purification Process of Recombinant Humanα1-Microglobulin by Pichia pastoris

作　　者：唐丹宁 罗安 王腾[1] 高建梅 蔡富强 温振国 贾兆君 TANG Danning;LUO An;WANG Teng;GAO Jianmei;CAI Fuqiang;WEN Zhenguo;JIA Zhaojun(Beijing Key Laboratory of Enze Biomass Fine Chemicals,College of New Materials and Chemical Engineering,Beijing Institute of Petrochemical Technology,Beijing 102617,China;Beijing Leadman Biochemistry Co.,Ltd.,Beijing 100176,China)

机构地区：[1]北京石油化工学院新材料与化工学院,恩泽生物质精细化工北京市重点实验室,北京102617 [2]北京利德曼生化股份有限公司,北京100176

出　　处：《中国生物工程杂志》2024年第4期33-42,共10页China Biotechnology

基　　金：北京市教育委员会科学研究计划(KM202210017010、KZ202210017025)资助项目。

摘　　要：目的:对肾脏损伤指标蛋白人α1-微球蛋白(alpha 1-microglobulin,A1M)的巴斯德毕赤酵母(Pichia pastoris)发酵与纯化工艺进行优化,实现高纯度、高亲和性重组A1M的克级制备。方法:利用1 L摇瓶培养体系单因素实验优化A1M发酵最适pH,随后进行5 L规模化高密度发酵,优化关键补料点及发酵时间;根据A1M抗原特性建立蛋白纯化工艺并进行N-糖基化鉴定和纯度鉴定;制备A1M免疫亲和柱并进行载量评估。结果:重组人A1M毕赤酵母摇瓶发酵最适初始pH 6.0,1 L摇瓶培养体系中表达水平达888 mg/L,且糖基化程度相对最低;5 L规模发酵的48~120 h采用溶氧(dissolved oxygen,DO)≥30%反向关联甲醇补料4~6 mL/(h·L),可有效控制DO核心区>20%;发酵48、72和96 h为关键补料点,表达水平为12.5 g/L,发酵58~78 h时空收率可达160~210 mg/(h·L),5 L罐发酵120 h批量达49 g;选用超滤浓缩、阳离子交换层析和脱盐层析柱建立A1M纯化工艺,总收率为18%;A1M抗原亲和柱偶联率达96%,偶联密度为2.46 mg/mL,对A1M绵羊多抗的载量为37 mg/mL。结论:成功建立重组人A1M抗原5 L规模的酵母发酵及纯化工艺,实现高纯度并具有免疫亲和力的A1M抗原的克级制备。Objective:To optimize the fermentation and purification process of humanα1-microglobulin(A1M),an index protein of kidney injury,in order to prepare recombinant A1M with high purity and high affinity in gram scale.Methods:The optimal pH of A1M fermentation was optimized by a single factor experiment in a 1 L shake flask culture system,followed by a 5 L large-scale fermentation,and the key feeding points and fermentation time were optimized.According to the characteristics of A1M antigen,the protein purification process was established,and N-glycosylation and purity identification were carried out.Finally,A1M immunoaffinity column was prepared and its loading was evaluated.Results:The optimal initial pH of human recombinant A1M Pichia pastoris was 6.0,the expression level reached 888 mg/L in 1 L shake flask culture system,and the degree of glycosylation was relatively lowest.During the 48~120 h of 5 L scale fermentation,DO≥30%reverse correlation methanol was used to feed 4~6 mL/(h·L-1),which can effectively control the core area of DO>20%.The 48th,72nd and 96th hours of fermentation were the key feeding points,and the expression level was 12.5 g/L.The space-time yield of fermentation from 58th to 78th hours was 160~210 mg/(L·h),and the batch of fermentation in a 5-L tank for 120 h was 49 g.The purification process of A1M was established by ultrafiltration concentration,cation exchange chromatography and desalting chromatography column,and the total yield was 18%.The coupling rate of A1M antigen affinity column was 96%,the coupling density was 2.46 mg/mL,and the loading capacity of A1M sheep polyclonal antibody was 37 mg/mL.Conclusion:The yeast fermentation and purification process of recombinant human A1M antigen in 5 L scale was successfully established,and the high purity and high affinity A1M antigen in gram scale was prepared.

关 键 词：Α1-微球蛋白 毕赤酵母 高密度发酵 糖基化修饰 

分 类 号：Q815[生物学—生物工程]