Tang S J, Sun K H, Sun G H, Chang T Y, Lee G C
Institute of Marine Biotechnology, Keelung, 20224, Taiwan.
Protein Expr Purif. 2000 Nov;20(2):308-13. doi: 10.1006/prep.2000.1304.
It is difficult to express recombinant Candida rugosa lipases (CRLs) in heterologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG for leucine. In this study, recombinant LIP4 in which all 19 CUG codons had been converted to a universal serine codon was overexpressed in Escherichia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion bodies and showed a low catalytic activity. To increase the amount of soluble form and activity of recombinant LIP4, the DNA was fused to the gene for thioredoxin (TrxFus-LIP4) and then expressed in E. coli strain AD494(DE3). This strategy promotes the formation of disulfide bonds in the cytosol and yields enzymatically active forms of LIP4. The purified recombinant TrxFus-LIP4 and LIP4 expressed in AD494(DE3) had the same catalytic profiles. In addition, recombinant LIP4 had higher esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein, and olive oil. This system for the expression of fungal lipase in E. coli strain AD494(DE3) is reliable and may produce enzymatically active forms of recombinant lipase without an in vitro refolding procedure.
在异源系统中表达重组皱褶假丝酵母脂肪酶（CRLs）很困难，因为皱褶假丝酵母使用非通用的丝氨酸密码子CUG编码亮氨酸。在本研究中，所有19个CUG密码子都已转换为通用丝氨酸密码子的重组LIP4在大肠杆菌BL21(DE3)中过表达。重组LIP4主要存在于包涵体中，且催化活性较低。为了增加重组LIP4的可溶性形式的量和活性，将DNA与硫氧还蛋白基因融合（TrxFus-LIP4），然后在大肠杆菌AD494(DE3)菌株中表达。该策略促进了胞质溶胶中二硫键的形成，并产生了具有酶活性的LIP4形式。在AD494(DE3)中表达并纯化的重组TrxFus-LIP4和LIP4具有相同的催化特性。此外，重组LIP4对长链酯具有较高的酯酶活性，而对三丁酸甘油酯、三油酸甘油酯和橄榄油的脂肪酶活性较低。在大肠杆菌AD494(DE3)菌株中表达真菌脂肪酶的这个系统是可靠的，并且可能无需体外重折叠程序就能产生具有酶活性的重组脂肪酶形式。
