[Efficient expression and purification of human beta-defensin-2 in E.coli].
To demonstrate the possibility of high-level expression of bioactive human beta-defensin-2 (hBD2) in E.coli, and to purify the recombinant hBD2.



DNA fragment containing mature hBD2 coding region (smhBD2-cDNA) was amplified by PCR, multiple copies of smhBD2-cDNA were linked using Bgl II and BamH I enzymes, pET32-nsmHBD2-cDNA with 1, 2, 4, or 8 copies of smhBD2-cDNA was constructed. The soluble and insoluble hBD2 proteins were separated and analyzed by SDS-PAGE analysis. The soluble protein underwent a separation process containing affinity chromatography, enterokinase digestion and ion exchange chromatography to get the recombinant hBD2 peptide. The bioactivity of recombinant hBD2 was examined by bacteria-inhibition tests in liquid culture.



The plasmids pET32-nsmHBD2-cDNA with 1, 2, 4 copies of smhBD2-cDNA were constructed and the expressed soluble protein accounted for 52 %, 48 %, and 31 % respectively. The plasmids with 8 copies expressed mainly insoluble protein with few in soluble form. The growth of E.coli K12D31 was dramatically suppressed with a inhibition rate of 90 %, when the final concentration of recombinant hBD2 reached between 0.4 to 0.5 mug/ml.



Fusion expression of human beta-defensin-2 with multiple joined genes in E.coli could increase the expression of hBD2.
Zhong
Zhi-xia
ZX
Institute of Bioengineering, Zhejiang University, Hangzhou 310027, China.
Ruan
Hong
H
Fan
Li-mei
LM
D000890:Anti-Infective Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
D000890:Anti-Infective Agents; C106641:DEFB4A protein, human; D011994:Recombinant Proteins; D023083:beta-Defensins