Efficient expression and purification of human beta-defensin-2 in E.coli / 浙江大学学报·医学版
Journal of Zhejiang University. Medical sciences
; (6): 585-589, 2006.
Article
em Zh
| WPRIM
| ID: wpr-332099
Biblioteca responsável:
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>
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Base de dados:
WPRIM
Assunto principal:
Farmacologia
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Proteínas Recombinantes
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Testes de Sensibilidade Microbiana
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Expressão Gênica
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Reação em Cadeia da Polimerase
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Cromatografia de Afinidade
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Beta-Defensinas
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Eletroforese em Gel de Poliacrilamida
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Escherichia coli
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Genética
Limite:
Humans
Idioma:
Zh
Revista:
Journal of Zhejiang University. Medical sciences
Ano de publicação:
2006
Tipo de documento:
Article