Cloning, expression and characterization of Kunming mice lactoferrin and its N-lobe.
The lactoferrin cDNA of Kunming mice was isolated by reverse transcription polymerase chain reaction and cloned into vector pET28a(+). Its deduced amino acid sequence was analyzed and compared with lactoferrin of other species. Its secondary and tertiary structure are predicted and modeled by bioinformatics tools online. Then recombinant Kunming mice lactoferrin and its N-lobe were both expressed successfully in the Escherichia coli BL21(DE3) in the form of inclusion bodies. After purification with Ni-NTA His-Bind resin, the yield of recombinant lactoferrin was 17 mg l(-1) with purity of 92.1%, and that of lactoferrin N-lobe was 20 mg l(-1) with purity of 98.5%. The inhibition efficiency of refolded lactoferrin N-lobe against Staphylococcus aureus ATCC 25923 reaches 48.6% at the concentration of 25 micromol l(-1). However, the refolded lactoferrin (12.5 micromol l(-1)) didn't display obvious inhibition activity in the test. The expression of recombinant Kunming mice lactoferrin and its N-lobe will be helpful for the study of lactoferrin on structure, function and application in a mouse model system.
Key Laboratory of Feed Biotechnology, Ministry of Agriculture, 100081 Beijing, China.
Jiarong
J
Wang
Zigang
Z
Tian
Da
D
Teng
Yalin
Y
Yang
Jiancheng
J
Hu
Jianhua
J
Wang
D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D007781:Lactoferrin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D008826:Microbial Sensitivity Tests; D023421:Models, Animal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000187:drug effects
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007781:Lactoferrin