Song Jung Min, An Young Jun, Kang Mee Hye, Lee Youn-Ho, Cha Sun-Shin
Marine Biology and Living Resources Research Department, Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea.
Protein Expr Purif. 2012 Apr;82(2):297-301. doi: 10.1016/j.pep.2012.01.020. Epub 2012 Feb 8.
Protein expression in Escherichia coli at 15-25°C is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6-10°C without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1l culture grown at 6 and 10°C, respectively. Cultivation at 10°C also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21(DE3). Consequently, E. coli cultivation at 6-10°C is an effective strategy for overcoming a major hurdle of the inclusion body formation.
在15 - 25°C的温度下于大肠杆菌中进行蛋白质表达被广泛用于提高重组蛋白的溶解度。然而，许多重组蛋白即使在这些低温条件下也会以不溶性形式表达。在此，我们表明，无需冷适应伴侣系统，只需将温度简单降低至6 - 10°C，重组蛋白就能以可溶性形式表达。通过使用大肠杆菌Rosetta - gami2(DE3)，我们分别从在6°C和10°C下培养的1升培养物中获得了1.8毫克和0.9毫克的南极隐虾甘露聚糖酶（CaMan）和纤维素酶（CaCel）。在10°C下培养还使得EM3L7（一种从宏基因组文库中分离出的脂肪酶）在大肠杆菌BL21(DE3)中以可溶性形式成功表达。因此，在6 - 10°C下培养大肠杆菌是克服包涵体形成这一主要障碍的有效策略。
