Recombinant production of cyanovirin-N, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium.
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-terminal sequencing. 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.
Mori
T
T
Laboratory of Drug Discovery Research and Development, Division of Cancer Treatment, Diagnosis and Centers, National Cancer Institute-FCRDC, Frederick, Maryland 21702-1201, USA.
Gustafson
K R
KR
Pannell
L K
LK
Shoemaker
R H
RH
Wu
L
L
McMahon
J B
JB
Boyd
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D019380:Anti-HIV Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001681:Biological Assay; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; C107733:cyanovirin N