Mori,T.; Gustafson,K.R.; Pannell,L.K.; Shoemaker,R.H.; Wu,L.; McMahon,J.B.; Boyd,M.R.: Protein Expr.Purif. 12: 151-18 (1998).
Abstract:
Here we describe the recombinant production and purification of a novel
anti-human immunodeficiency virus (HIV) protein, cyanovirin-N (CV-N),
in Escherichia coli. Initial attempts to express CV-N using a vector
containing an ompA signal peptide sequence resulted in production of an
intractable mixture of the full-length (101 amino acid residue) protein
and a truncated form lacking the first two N-terminal amino acids. The
truncated protein was observed regardless of the host cell line, culture
conditions, or induction time. These observations suggested that an as yet
unidentified protease or peptidase was responsible for proteolytic
cleavage between the second and third N-terminal amino acids of CV-N when
presented as an ompA-CV-N fusion protein. When the ompA signal peptide
sequence was replaced by a pelB signal peptide sequence, CV-N was produced
in high yield as a single, homogeneous protein. This was confirmed by
electrospray ionization mass spectrometry and N-terminalsequencing. This
expression system provides a basis for large-scale production of clinical
grade CV-N for further research and development as an anti-HIV
microbicide.
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