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 Human α1-Microglobulin by Pichia pastoris
唐丹宁 1罗安 1王腾 1高建梅 2蔡富强 2温振国 1贾兆君1
作者信息
- 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
- 折叠
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.
Abstract
Objective:To optimize the fermentation and purification process of human α1-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 A1 M 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Key words
α1-Microglobuli/Pichia pastori/High density fermentatio/Glycosylation modification引用本文复制引用
基金项目
北京市教育委员会科学研究计划(KM202210017010)
北京市教育委员会科学研究计划(KZ202210017025)
北京石油化工学院青年教师科技创新资助项目(15031862005/321)
北京利德曼生化股份有限公司对本研究的支持()
出版年
2024
NETL
NSTL
万方数据