Polyhedrin initiator codon altered to AUU yields unexpected fusion protein from a baculovirus vector.
A recombinant baculovirus expression vector was constructed to express the core (capsid) protein of the hepatitis B virus. Along with the expected 21-kDa polypeptide, a second 24-kDa protein was observed. Immunoprecipitation and immunoblotting using a rabbit polyclonal anticore antiserum demonstrated that the two proteins were related. The core gene originally was cloned in-frame with the polyhedrin initiator codon that had been altered to AUU as a means of preventing fusion protein formation. A transient expression assay revealed expression of the 24-kDa protein was prevented if a frame-shift mutation was created upstream of the HBV core translation start site. These results suggest that the 24-kDa protein was the result of an unexpectedly high level of translation initiation at the AUU codon that gave rise to a polyhedrin-HBV core fusion protein. The 24-kDa core protein was shown to be a polyhedrin fusion protein by immunoblotting with an antipolyhedrin antiserum, and initiation at the AUU was demonstrated by amino terminal protein sequencing. Methods to prevent undesired fusion protein expression using this or similar vectors are discussed.
Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, TX 78228-0147.
B
B
Beames
S
S
Braunagel
M D
MD
Summers
R E
RE
Lanford
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003062:Codon; D004279:DNA, Viral; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D000077183:Occlusion Body Matrix Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014758:Viral Core Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*; D015678:Viral Structural Proteins
D003062:Codon; D004279:DNA, Viral; D000077183:Occlusion Body Matrix Proteins; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus