整体优化的信号肽和人溶菌酶基因在毕赤酵母的高效表达  

De novo design of α-factor signal and human lysozyme gene leads to highlevel expression in Pichia pastoris

作　　者：陈珊珊[1] 丁健[1] 李鑫 刘军 贾禄强 槐强强 孙佼文 史仲平[1] 

机构地区：[1]江南大学生物工程学院/工业生物技术教育部重点实验室,江苏无锡214100 [2]武汉轻工大学生物与制药工程学院,湖北武汉430040

出　　处：《江苏农业学报》2018年第1期20-28,共9页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(21606106);江苏省自然科学基金项目(BK20150127、BK20160162)

摘　　要：人源溶菌酶作为一种天然的抑菌活力物质,在畜牧、医药及食品等行业具有潜在的应用价值。本研究将信号肽序列和人源溶菌酶基因碱基序列作为一个整体进行密码子优化。并在优化的信号肽序列2处位置,共新插入39个碱基,构成增长的优化信号肽+溶菌酶优化基因。将2种优化的信号肽+溶菌酶基因碱基序列分别插入pPICZαA载体上,并整合到毕赤酵母KM71的基因组上,获得重组子K1(整体优化的α信号肽+人源溶菌酶基因)和K4(整体优化的α信号肽+人源溶菌酶基因又整合了增长信号肽)。摇瓶诱导表达72 h后,离心取上清液,通过Bradford法和比浊法测定发现,K4的蛋白质表达量和酶活力均高于K1。5 L发酵罐下,采用高密度诱导表达策略,诱导54 h后K4的总蛋白质表达量可达3.02 g/L,所表达的人源溶菌酶活力达到324 072.0 U/ml。表明整体优化的α信号肽+人源溶菌酶基因又整合了增长信号肽有利于提高溶菌酶的分泌效率,实现高效表达。Human lysozyme( hLYZ) has potential application value in many fields,as a natural antibacterial agent.In this study,the codons of α-factor signal and hLYZ gene were optimized as a whole by using a de novo design strategy.Based on the optimization,39 nucleotides were inserted into two different locations of α-factor signal sequence,obtaining an extended α-factor signal followed with hLYZ gene. These two optimized sequences were chemically synthesized and respectively constructed into the yeast secretory expression vector pPICZαA,then transformed into Pichia pastoris KM71 by electroporation. Two series of recombinants were harvested,the optimized α-factor+hLYZ( named K1) and the extended optimized α-factor+hLYZ( named K4). In shake-flask culture,protein expression content and antibacterial activity of the recombinant K4 were higher than those of K1,and measured by Bradford assay and turbidimetric method,respectively.In 5 L fermentor,the protein expression level of K4 achieved the maximum of 3. 02 g/L at 54 h after induction by using the high cell density induction strategy,in which the antibacterial activity of the recombinant human lysozyme reached 324 072.0 U/ml. The results indicated that the extended α-factor signal could greatly promote secretion of the recombinant human lysozyme,leading to achievement of high-level expression.

关 键 词：人源溶菌酶 信号肽序列 毕赤酵母 高效表达 

分 类 号：Q814.9[生物学—生物工程]