Your browser doesn't support javascript.
loading
Efficient expression and purification of human beta-defensin-2 in E.coli / 浙江大学学报·医学版
Article de Zh | WPRIM | ID: wpr-332099
Bibliothèque responsable: WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To demonstrate the possibility of high-level expression of bioactive human beta-defensin-2 (hBD2) in E.coli, and to purify the recombinant hBD2.</p><p><b>METHODS</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>Fusion expression of human beta-defensin-2 with multiple joined genes in E.coli could increase the expression of hBD2.</p>
Sujet(s)
Texte intégral: 1 Indice: WPRIM Sujet Principal: Pharmacologie / Protéines recombinantes / Tests de sensibilité microbienne / Expression des gènes / Réaction de polymérisation en chaîne / Chromatographie d'affinité / Bêta-Défensines / Électrophorèse sur gel de polyacrylamide / Escherichia coli / Génétique Limites du sujet: Humans langue: Zh Texte intégral: Zhejiang Daxue xuebao. Yixue ban Année: 2006 Type: Article
Texte intégral: 1 Indice: WPRIM Sujet Principal: Pharmacologie / Protéines recombinantes / Tests de sensibilité microbienne / Expression des gènes / Réaction de polymérisation en chaîne / Chromatographie d'affinité / Bêta-Défensines / Électrophorèse sur gel de polyacrylamide / Escherichia coli / Génétique Limites du sujet: Humans langue: Zh Texte intégral: Zhejiang Daxue xuebao. Yixue ban Année: 2006 Type: Article