[Clone, construct, expression and verification of lactoferricin B gene and several sequence mutations in yeast].
OBJECTIVE
To construct the eucaryotic recombinant plasmid of pYES2/LactoferricinB expressing in yeast of S. cerevisiae, of which the expressed protein antibacterial activity was verified in preliminary.


METHODS
By self-template PCR method, the gene of Lactoferricin B and its several sequence mutations were amplified with the parts of the pre-synthesized single chains. And then Lactoferricin B gene and its mutants were cloned into the vector of pYES2 to construct the recombined expression plasmid pYES2/Lactoferricin B etc. extracted and used to transform the yeast S. cerevisiae. The expressions of proteins were determined after induced by galactose. The expression proteins were collected and purified by hydronium-exchange column, and the bacterial inhibited test was applied to identify the protein antibacterial activities.


RESULTS
The PCR amplifying and DNA sequencing tests indicated that the purpose plasmid contained the Lactoferricin B gene and several mutations. The induced target proteins were confirmed by SDS-PAGE electrophoresis and mass spectrum test. The protein antibacterial activities of mutations were verified in preliminary.


CONCLUSIONS
The recombined plasmid pYES2/Lactoferricin B etc. are successfully constructed and induced to express in yeast cell of S. cerevisiae; the obtained recombined protein of Lactoferricin B provides a basis for further research work on the biological function and antibacterial activity.
Feng
Yong-qian
YQ
Department of Biochemistry and Molecular Biology, West China School of Preclinicial and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Zha
Xiao-jun
XJ
Zhai
Chao-yang
CY
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D007781:Lactoferrin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004220:Disulfides; C082171:lactoferricin B; D007781:Lactoferrin