蛛丝蛋白MiSp重组模块R1R2CT表达及其纤维化动力学研究

Recombinant Expression and Fibrillisation Analysis of MiSp R1R2CT Module

为探索蛛丝蛋白模块单元在成丝过程中的作用,研究了大腹园蛛MiSpR1R2CT功能模块在不同pH条件下的纤维化动力学特性。大腹园蛛MiSp蛋白的R1R2CT功能模块与硫氧还蛋白融合,并在BL21（DE3）中进行表达,表达量约为15mg/LLB培养基。R1R2CT蛋白在pH7.5和6.5时较为稳定,在22h之内不发生纤维化;当pH值降至5.5时,R1R2CT在前2h内快速纤维化,3～5h趋势较为平缓;5h之后R1R2CT蛋白再次出现ThT信号的快速增长,7h后保持缓慢增长至22h。R1R2CT的增长速率及幅度高于单独的CT（C-termi-nal）蛋白,而单独的R1R2在pH7.5、6.5和5.5时均保持稳定,表明重复区依赖CT模块的快速纤维化模式。已有研究表明,CT在重复区纤维化过程中起晶核作用,该成果也从反面证实CT的晶核理论。

Fibrillisation dynamics under different pHs were analyzed for determination of the functions of spider silk protein modules in silk formation process. According to the complete MiSp gene sequence, RIR2CT fused with thioredoxin was expressed successfully via gene recombinant technology in BL21 （DE3）, which resulted in a fusion protein as thioredoxin-thrombin cleavage site-R1R2CT. The expression level of recombinant proteins is about 15 mg/L LB medium. R1R2CT maintains soluble conformation and no positive ThT signals were detected under pH 7.5 and 6.5. Enhanced ThT signals were collected when pH decreased to 5.5, and dur- ing the following 3-5 h increased much more smoothly. After the first five hours, second swiftly sharp ThT sig- nals appeared, and maintained to seven hours, after which the second relatively smooth increasement of ThT signals lasted all over the measurement time. The rate and latitude of R1R2CT ThT signal was higher than that of CT alone, and under pH 7.5, 6.5 and 5.5 no positive ThT signals were detected for R1R2 proteins indicated a quick fibrillisation process of R1R2 dependent on CT, and also CT is an essential part for spider silk forma- tion. The fibrillation of RIR2CT to some extent supports the nucleation theory in some other light.