大肠杆菌表达高性能融合鱿鱼环齿-类弹性蛋白的发酵优化  

Optimization of Fermentation Process for Production of Elastin-Like and Squid Ring Teeth Fusion Proteins for the Construction of Robust Fibers in Escherichia coli

作　　者：姜正日 殷仁凯 王泽建[1] 钱江潮[1] JIANG Zhengri;YIN Renkai;WANG Zejian;QIAN Jiangchao(State Key Laboratory of Bioreactor Engineering,School of Biotechnology,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学生物工程学院,生物反应器工程国家重点实验室,上海200237

出　　处：《华东理工大学学报（自然科学版）》2024年第1期71-79,共9页Journal of East China University of Science and Technology

基　　金：国家自然科学基金(21704099,21877104)。

摘　　要：为了实现重组蛋白SRT-ELP36在大肠杆菌中的高效表达,利用携带重组蛋白SRTELP36基因的pET-25b(+)表达载体,分别在摇瓶和5 L发酵罐上进行了发酵条件优化,并在50 L发酵罐中进行放大验证。经转化表达质粒后,宿主菌BL21(DE3)的菌体生长和蛋白体积产量均高于BLR(DE3),可用于蛋白SRT-ELP36表达。摇瓶发酵条件优化结果表明,TB(Terrific Broth)培养基、装液比(装液量与摇瓶体积之比)为5%、诱导温度为37℃、对数生长结束期添加0.5 mmol/L的IPTG(异丙基-β-D-硫代半乳糖苷)诱导剂,菌体最高OD_(600)(波长600 nm处的吸光值)达到22.3,最高蛋白体积产量达0.60 g/L。在5 L发酵罐中进行补料分批培养条件优化,在OD_(600)为35时添加终浓度为0.5 mmol/L的IPTG启动诱导,并控制过程菌体氧消耗速率(OUR)为(180±5) mmol/(L·h),菌体的最高OD_(600)和蛋白体积产量分别达到了88、1.85 g/L,单位菌体蛋白产量为70 mg/g。在50 L发酵罐中,参照5 L工艺控制条件进行发酵过程放大,菌体最高OD_(600)达到103,重组蛋白SRT-ELP36的体积产量和单位菌体产量进一步提高到了2.22 g/L和90 mg/g,是目前文献报道SRT蛋白表达的最高水平,为高性能融合鱿鱼环齿-类弹性蛋白的工业化生产提供了基础。The recombinant structural protein SRT-ELP36 consisted of repetitive squid ring teeth protein segment and cationic elastin-like polypeptide sequence(PAATAVSHTTHHAP-VPGVG(VPGKG)5)can be used to fabricate protein fibers with extraordinary mechanical properties and biocompatibility.In order to improve the production of recombinant protein SRT-ELP36,the pET-25b(+)vector carrying SRT-ELP36 expression cassette was transformed into Escherichia coli,and the fermentation process was optimized in shake flasks and bioreactors.In comparison with the recombinant E.coli BLR(DE3),BL21(DE3)grew more rapidly with a higher expression level of SRT-ELP36.Thus,batch cultivation of recombinant BL21(DE3)in shake flasks was optimized to achieve the highest cell density OD600 of 22.3 and SRT-ELP36 production of 0.60 g/L in TB(Terrific Broth)medium with 5%volume ratio of medium to flask after 0.5 mmol/L IPTG induction at 37℃at the end of exponential growth phase.Subsequently,the fed-batch fermentation process in a 5 L bioreactor was developed:induction with 0.5 mmol/L IPTG at the OD600 of 35,a high OUR level up to(180±5)mmol/(L·h).With the highest OD600 of 88,the highest volumetric and specific production of SRT-ELP36 of 1.85 g/L and 70 mg/g was obtained,respectively.Based on the cultivation conditions optimized in the 5 L bioreactor,high cell density fermentation of recombinant BL21(DE3)was successfully carried out in a 50 L bioreactor with the highest OD600 of 103.SRT-ELP36 volumetric and specific production were increased to 2.22 g/L and 90 mg/g,respectively,which was the highest expression level of Squid ring teeth protein as reported to date and providing the foundation for eventual application of SRT-ELP36 in industrial fabrication of mechanically strong fibers.

关 键 词：大肠杆菌 鱿鱼环齿蛋白 类弹性蛋白 力学性能 发酵优化 

分 类 号：Q815[生物学—生物工程]