【摘要】 通过对漆酶成熟肽编码序列的N端(G1n²-Val⁸⁴)及中部一富含GC碱基区域(Gly²⁷⁸-Pro³⁰³进行密码子优化,成功实现嗜热细菌嗜热栖热菌Thermus thermophilus HB27胞内产极耐热漆酶在大肠杆菌宿主内的超量表达,热激诱导表达后细胞提取液中漆酶活性从（20.7±1.5）U/L升高到（1790.2±81.6）U/L,重组蛋白表达水平提高了86倍。利用全蛋白的极端耐热性,采用两步纯化方法（85℃热预处理和疏水柱层析）,获得电泳纯的重组酶蛋白,比酶活为14 U/mg,最终收率为62%。这是首次报道采用pHsh热激表达系统实现该极耐热性细菌漆酶在大肠杆菌宿主体内的高效表达。更多还原

【Abstract】 A simple selective codon optimization strategy was devised to quickly improve the expression of a hyperthermo-philic laccase from Thermus thermophilus HB27(named as Tth-laccase) in E.coli using pHsh expression vector.After the region A,from amino acid 2(Gln) to amino acid 84(Val) and B,GC-rich area from amino acid 278(Gly) to amino acid 303(Pro) of Tth-laccase coding sequence were codon optimized through site-directed mutagenesis,the laccase activity in the cell extract after expression was increased from(20.7±1.5) U/L to(1 790.2±81.6) U/L.The protein expression level was increased by 86-fold.Taking advantage of the remarkable thermostability of holoenzyme,a simple two-step procedure including heat treatment and hydrophobic interaction chromatography resulted in the purified protein of14 U/mg with a yield of 62%.This is the first report on over expression of Tth-laccase in Escherichia coli by using pHsh expression system.更多还原