Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.
Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.
Chen
Gen-Hung
GH
Department of Cosmetic Science,, Providence University, 200 Chung-Chi Rd., Taichung 43301, Taiwan.
Yin
Li-Jung
LJ
Chiang
I-Hua
IH
Jiang
Shann-Tzong
ST
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D003257:Consumer Product Safety; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006041:Goats; D006639:Histidine; D006801:Humans; D007781:Lactoferrin / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D013696:Temperature; D013997:Time Factors
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C082171:lactoferricin B; D006639:Histidine; D007781:Lactoferrin