High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens.
Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively.
Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, CAS, Hunan 410125, China.
Zhiru
Z
Tang
Youming
Y
Zhang
Adrian Francis
AF
Stewart
Meimei
M
Geng
Xiangsha
X
Tang
Qiang
Q
Tu
Yulong
Y
Yin
D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001089:Arabinose / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D007781:Lactoferrin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007782:Lactoglobulins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020637:Photorhabdus / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D000900:Anti-Bacterial Agents; D007782:Lactoglobulins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C502040:lactoferrampin; C082171:lactoferricin B; D001089:Arabinose; D007781:Lactoferrin