Efficient synthesis of a 72-kDa mitochondrial polypeptide using the yeast Ty expression system.
Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBgIII expression vector was initially modified for this purpose by inserting the factor X(a) protease cleavage site. The TyA gene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryotic yst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungus Podospora anserina, were subsequently fused to the factor X(a) cleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor X(a) cleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor X(a) treatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.
Pöggeler
S
S
Lehrstuhl für Allgemeine Botanik, Ruhr-Universität Bochum, Germany.
Schwerk
C
C
Kämper
U
U
Kück
U
U
D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli; D015951:Factor Xa / Q000096:biosynthesis; D005800:Genes, Fungal; D015151:Immunoblotting; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D018626:Retroelements; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013098:Xylariales / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D018626:Retroelements; D015951:Factor Xa; D014448:Tyrosine-tRNA Ligase