High-level expression of housefly cecropin A in Escherichia coli using a fusion protein

Xueli Zheng; Wei Wang

摘要

Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Musca domestica (M. domestica) cecropin in Escherichia coli (E. coli) and to identify the expressed products. Methods:The genomic sequence of M. domestica cecropin A (MC) and M. domestica ubiquitin (UBI) were searched from Genbank and amplified by reverse transcriptase polymerase chain reaction (RT-PCR). Two expression plasmids, pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E. coli and were then induced by Isopropylβ-D-1-Thiogalactopyranoside (IPTG). The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fusion protein Trx-MC was verified by Western blot analysis. The bactericidal activity of the purified MC was quantitatively determined using E. coli BL21(DE3). Results:The result showed that the fusion proteins were successively expressed in E. coli BL21 cells. A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained, and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E. coli. MC exhibited antimicrobial activity. Conclusions:With high-level expression of housefly cecropin A in E. coli using a fusion protein, MC exhibited antimicrobial activity.

机译：目的：研究利用分子伴侣对家蝇（M. domestica）cecropin在大肠杆菌（E. coli）中高水平表达的影响，并鉴定表达产物。方法：从Genbank中检索家蝇天蚕素A（MC）和家蝇泛素（UBI）的基因组序列，并通过逆转录聚合酶链反应（RT-PCR）进行扩增。构建两个表达质粒pET32a-MC和pET32a-UBI-MC并转移到大肠杆菌中，然后用异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）诱导。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析融合蛋白Trx-MC和Trx-UBI-MC的表达。融合蛋白Trx-MC通过蛋白质印迹分析证实。使用大肠杆菌BL21（DE3）定量测定纯化MC的杀菌活性。结果：结果表明融合蛋白在大肠杆菌BL21细胞中连续表达。阳性染色的代表Trx-MC靶蛋白的24 kDa的条带被阳性染色，并且在预期的位置也观察到代表成熟MC蛋白水解的4 kDa的条带。 Trx-UBI-MC在大肠杆菌中的表达水平高于Trx-MC。 MC表现出抗菌活性。结论：MC通过融合蛋白在大肠杆菌中高表达家蝇天蚕素A，具有抗菌活性。

High-level expression of housefly cecropin A in Escherichia coli using a fusion protein

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