破菌时可自动降解宿主核酸的大肠杆菌BL21(DE3)的lpxM突变株构建

Constructing Modified Protein-producing Escherichia coli Capable of Autohydrolysing Host Nucleic Acid During Cell Lysis

摘要:

研究利用Red同源重组技术对常用大肠杆菌表达宿主菌BL21(DE3)进行改良, 构建破菌时可自动降解宿主核酸的大肠杆菌表达宿主菌, 该菌株可望有助于解决因破菌时宿主菌染色体核酸释放给后续纯化重组蛋白工作带来的困难。将N端连有OmpA的信号肽的S. aureus nucleaseB(nucB)表达框整合至E. coli BL21(DE3)的lpxM位点, 改造后菌株(称为BLN)经诱导能表达nucB、并分泌至周质空间, 这样可使宿主核酸免受该酶“毒性”影响, 菌体裂解后, nucB释放,能自动降解宿主核酸。BLN菌体生长状态以及表达外源重组蛋白的能力与出发菌基本一致。

Abstract:

We used Red recombination system to construct a modified E. coli protein-producing strain capable of autohydrolysing host nucleic acid. E. coli BL21(DE3), a common protein-producing strain, was used as starting material. The modified E. coli expres sion host had a staphylococcal nuclease expression cassette within the host chromosome lpxM locus. The Staphylococcus aureus nuclease was expressed as a fusion to the ompA signal peptide, and was translocated to the periplasm of the cell, protecting the host nucleic acid from the toxic activity during growth. The nuclease was released during cell lysis and subsequently hydrolyzed host nucleic acid in the lysate. Results show that the modified strain had sufficient nuclease activity to completely autohydrolyze the host chromosomal DNA and produced same amount of recombinant proteins as the original strain.