Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.
Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.
Nilsson
B
B
Abrahmsén
L
L
Uhlén
M
M
D000469:Alkaline Phosphatase / Q000235:genetics / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000302:isolation & purification*; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D010957:Plasmids; D012685:Sepharose / Q000031:analogs & derivatives; D013205:Staphylococcal Protein A / Q000235:genetics*; D013210:Staphylococcus / Q000201:enzymology / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification*
D004800:Enzymes, Immobilized; D013205:Staphylococcal Protein A; C036210:immunoglobulin G-sepharose; D012685:Sepharose; D004262:DNA Restriction Enzymes; D000469:Alkaline Phosphatase; D005696:Galactosidases; D001616:beta-Galactosidase