O'Neill G P, Kilburn D G, Warren R A, Miller R C
Appl Environ Microbiol. 1986 Oct;52(4):737-43. doi: 10.1128/aem.52.4.737-743.1986.
A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.
一种重组外切葡聚糖酶在大肠杆菌中表达，其表达水平超过细胞总蛋白的20%。为达到这种高水平的过量表达，将外切葡聚糖酶基因编码序列与一个合成核糖体结合位点、一个起始ATG融合，并置于失控复制质粒载体pCP3（E.雷马特、H.曹和W.菲尔斯，《基因》22:103 - 113，1983年）上噬菌体λ左向启动子的控制之下。除了在起始ATG附近插入了一个天冬酰胺外，高表达的外切葡聚糖酶与天然外切葡聚糖酶相同。过量产生的外切葡聚糖酶能够很容易地以富集形式作为不溶性聚集体分离出来，并且通过将聚集体溶解于6M尿素或5M盐酸胍中可恢复外切葡聚糖酶活性。由于外切葡聚糖酶基因的密码子使用与大肠杆菌基因的密码子使用差异如此显著，外切葡聚糖酶在大肠杆菌中的过量表达表明密码子使用可能不是该生物体中异源基因表达的主要障碍。
