Cloning, expression and purification of the immunity factor associated with leucocin A production.
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Date
2004
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Abstract
Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22.
Bacteriocin producer strains possess an immunity protein, which enables the strain to
protect itself against its own bacteriocin. The immunity gene from Leucoconostoc
gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was
cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The
resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain
JM103. The clone putative, was confirmed by DNA sequencing and southern blot
hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of
3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein
expressed within E. coli. The fusion construct was isolated by affinity chromatography.
Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium
UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a
C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the
stability of the immunity gene fusion protein were compared using a Biacore 2000. The
supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22
were tested for interaction with the fusion construct. Surface Plasmon resonance studies
indicated that there was no binding partner present in the supernatant which would
influence the immunity process. However, a stable interaction was found between the
immunity protein and an orphan ligand within the cytoplasm.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.
Keywords
Bacteriocins., Leucoconostoc gelidium., Proteins., Immunogenetics., Immunity., Lactic acid bacteria., Theses--Genetics.