Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.
A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.
Smith
G R
GR
David North Plant Research Centre, Bureau of Sugar Experiment Stations, Brisbane, Queensland, Australia.
Ford
R
R
Bryant
J D
JD
Gambley
R L
RL
McGhie
T K
TK
Harding
R M
RM
Dale
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000276:immunology*; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D008969:Molecular Sequence Data; D010945:Plants, Edible / Q000382:microbiology; D017800:Potyvirus / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins
D000914:Antibodies, Viral; D017931:DNA Primers; D011993:Recombinant Fusion Proteins