Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

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Abstract
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.