基于T7转录系统的谷氨酸棒杆菌重组蛋白高效表达系统的创建  

Creation of the efficient recombinant protein expression system based on the T7 transcription system in Corynebacterium glutamicum

作　　者：任晓昕 韩琳琳 武子淇 谷敏 徐大庆[1] REN Xiaoxin;HAN Linlin;WU Ziqi;GU Min;XU Daqing(College of Life Sciences,Hebei Agricultural University,Baoding 071000,China;Li County Agricultural and Rural Bureau of Hebei Province,Baoding 071400,China)

机构地区：[1]河北农业大学生命科学学院,河北保定071000 [2]河北省蠡县农业农村局,河北保定071400

出　　处：《河北农业大学学报》2023年第4期91-97,共7页Journal of Hebei Agricultural University

基　　金：河北省自然科学基金(C2020204085);河北省重点研发计划项目(22326610D).

摘　　要：本研究以前期构建的大肠杆菌-谷氨酸棒杆菌穿梭载体pAU2为基础,通过添加T7 RNA聚合酶编码基因及人工合成的克隆表达区,构建了1个新的谷氨酸棒杆菌分泌型基因高效表达载体pAU29KS。pAU29KS载体克隆/表达盒使用T7启动子作为目的基因的启动子,通过载体序列中T7 gene 1基因编码的T7 RNA聚合酶与T7启动子互作来进行目的基因的高效转录;启动子区下游使用谷氨酸棒杆菌核糖体结合位点RBS保守序列(gaaagga)来高效起始目的蛋白合成;克隆/表达盒RBS与多克隆位点MCS之间使用谷氨酸棒杆菌强信号肽cgl_2070编码序列来进行目的蛋白的胞外高效分泌。以嗜热脂肪土芽孢杆菌的α-淀粉酶AmyF作为报告蛋白,进行谷氨酸棒杆菌C.glutamicum/pAU29KS表达系统的蛋白分泌生产能力检测。透明圈法检测结果显示,工程菌株C.glutamicum/pAU29KS-amyF能够在淀粉平板上产生清晰可见的透明圈;培养物上清液的SDS-PAGE考马斯亮蓝染色和Western Blotting检测结果都显示出清晰的特异性条带,与AmyF预测分子量相一致;淀粉酶活性检测结果显示,培养物上清液呈现高淀粉酶活力,而细胞裂解上清液未检测到淀粉酶活力,说明高效表达的α-淀粉酶在强信号肽cgl_2070的介导下完全分泌到胞外培养基中。本研究构建的基于T7转录系统的C.glutamicum/pAU29KS能够对目标蛋白进行高效分泌生产,是一套新的谷氨酸棒杆菌分泌型重组蛋白表达系统。In this study,a new efficient secretion-type gene expression vector pAU29KS was constructed by sequentially adding the T7 RNA polymerase-encoding gene and synthetic cloning/expression cassette to the E.coli-C.glutamicum shuttle vector pAU2 constructed in our previous work in C.glutamicum.The pAU29KS vector employs the T7 promoter that the target gene can be efficiently transcribed by interaction between the T7 RNA polymerase and the T7 promoter.The conserved RBS sequence(gaaagga)of C.glutamicum was located downstream of the promoter region to efficiently initiate target protein synthesis.The strong signal peptide cgl_2070-encoding sequence of C.glutamicum between RBS and the multiple cloning sites(MCS)was used to mediate efficient extracellular secretion of the target protein.Using the alpha-amylase of Geobacillus stearothermophilus as a reporter protein,the ability to protein secretory production of the C.glutamicum/pAU29KS expression system was tested.The results of transparent circle method showed that there were appeared clear transparent circles around the strain C.glutamicum/PAU29KS-amyF cultures on the starch plate.The SDS-PAGE staining and Western Blotting results of the supernatants of the strain C.glutamicum/PAU29KS-amyF culture showed clear specific AmyF bands.The tests of AmyF activities demonstrated that the amylase activity was high in the supernatant of the strain C.glutamicum/PAU29KS-amyF culture,while no amylase activity was detected in the supernatant of the strain C.glutamicum/PAU29KS-amyF cell lysis,suggesting that the efficiently expressed alpha-amylase was completely secreted into the medium.These results demonstrated that the C.glutamicum/pAU29KS system can efficiently express and secrete the target protein,and is an excellent secretion-type C.glutamicum recombinant proteins production system.

关 键 词：谷氨酸棒状杆菌 T7转录系统 重组蛋白表达和分泌 

分 类 号：Q71[生物学—分子生物学]