一种新的产生天然蛋白的不依赖于连接的克隆载体

报道一种新的不依赖于连接的克隆（LIC）载体．该载体被缺刻的内切酶N．BbrCIA和限制性内切酶SwaI作用产生长的粘性末端．靶向基因的PCR片段在4mmol／LdGTP的孵育下被T4DNA聚合酶处理生成互补粘性末端．将处理过的载体和PCR产物共转化到大肠杆菌体内，插入载体的接合能够被宿主酶和给定的重组质粒修复．接着引入到大肠杆菌表达系统，该融合蛋白能够被表达和纯化，再用烟草蚀刻病毒TEV蛋白酶处理将移去靶蛋白的N末端，产生天然蛋白．两个基因，绿色荧光蛋白基因egfp和硫氧环蛋白基因txn已被用于这个系统．结果表明LIC策略对于高通量克隆和表达是高效的，对于天然蛋白的产生也同样高效．

Novel ligation-independent cloning vectors for production of native proteins

A new strategy of ligation-independent cloning was reported in this paper. These ligation-independent cloning vectors were digested with nicking endonuclease N. BbvCIA and restriction endonuclease Swa I to generate specific long sticky ends. PCR fragments of target genes were treated with T4 DNA polymerase in the presenting of 4 mmol/L dGTP to form complementary cohesive ends. Upon co-transformation of treated vectors and PCR products into Escherichia coli, the vector insert junction would be repaired by host enzymes and given recombinant plasmids. After introduced into Escherichia coli expression system, fusion proteins can be expressed and purified. Following treatment with TEV protease will remove all extra amino acids at the N-terminal of the target proteins and yield native proteins. Two genes, green fluorescence protein（egfp） and thioredoxin（txn）, were used to validate this system. The LIC strategy is efficient for high-throughput cloning and expression, as well as generating of native proteins.