Nucleic acid construct for endogenously expressing RNA polymerase in cells

• Main Authors: XUE YINGE, CHAI ZHI, YU XUE, DAI TIANCHUN, LIU SHUAILONG, GUO MIN, LI HAIYANG, WANG HAIPENG
• Format: Patent
• Language: Chinese; English
• Published: 05.03.2019
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

The invention provides a nucleic acid construct for endogenously expressing RNA polymerase in cells, and particularly discovers a novel nucleic acid construct capable of greatly enhancing protein translation efficiency. The nucleic acid construct consists of a promoter with specific promotion intensity (such as RNR2, ADH1, GAPDH, TEF1, PGK1 and SED1) and coding sequences of proteins of RNAP (suchas T7RNAP, T3RNAP, T4RNAP and T5RNAP), if the nucleic acid construct of the invention is applied into a yeast extracorporeal protein synthesis system, the activity of synthesized luciferase is quite high relative to light unit value (RLU), and the effect can be the same as the effect of T7RNAP added exogenously. The effect can reach up to 6.6* 107. 本发明提供了种细胞中内源性表达 RNA 聚合酶的核酸构建物，具体地，发现了种可大幅度增强蛋白质翻译效率的新型核酸构建物，本发明的核酸构建物由具有特定启动强度的启动子（如RNR2、ADH1、GAPDH、TEF1、PGK1、SED1等）和RNAP（如T7RNAP、T3RNAP、T4RNAP、T5RNAP等）蛋白的编码序列构成，在酵母体外蛋白质合成体系中应用本发明的核酸构建物，所合成的荧光素酶活性的相对光单位值（RLU）非常高，可达到与外源加入T7RNAP相同效果。可高达6.6×10。