Production of bioactive human alpha-defensin 5 in Pichia pastoris.
Human alpha-defensin 5 (HD5), a small cationic peptide, is expressed in Paneth cell granules of small intestinal crypts. HD5 exhibits high antimicrobial activity against a broad spectrum of pathogenic agents, including bacteria, fungi, and viruses. In this study, the constitutive expression of HD5 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris (P. pastoris). HD5 cDNA was amplified by polymerase chain reaction (PCR) using human lung cell cDNA as template. The 96-bp DNA fragment encoding mature HD5 peptide (amino acid 63-94) was subcloned into the yeast expression vector and transfected into P. pastoris X-33 expression host by electroporation. The recombinant HD5 (rHD5) was detected in the supernatant of transfected yeast by western blot analysis. The recombinant HD5 crude extract from transfected P. pastoris showed antimicrobial activities against Salmonella typhimurium, Staphylococcus aureus and pathogenic E. coli. However, rHD5 did not inhibit the growth of lactic acid bacteria such as Lactobacillus bulgaricus, Bifidobacterium bifidum, or B. longum. These results indicated that the rHD5 expressed in P. pastoris selectively inhibited the growth of specific bacteria.
Hsu
Kung-Hao
KH
Department of Animal Science and Biotechnology, Tunghai University, Taichung, Taiwan, R.O.C.
Pei
Chi
C
Yeh
Jan-Ying
JY
Shih
Chung-Hsung
CH
Chung
Yun-Chin
YC
Hung
Lien-Tsung
LT
Ou
Bor-Rung
BR
D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005658:Fungi / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D019879:Paneth Cells; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D023084:alpha-Defensins / Q000235:genetics / Q000378:metabolism*
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; C500225:DEFA5 protein, human; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D023084:alpha-Defensins