Carbohydrate-recognition domains as tools for rapid purification of recombinant eukaryotic proteins.
Methods have been developed for expression and purification of eukaryotic proteins by creating fusions with the carbohydrate-recognition domain (CRD) of the galactose-specific rat hepatic lectin. In order to generate the fusion proteins, vectors have been constructed so that cDNAs for passenger proteins can be inserted in any reading frame following a segment of DNA encoding the CRD. The feasibility of using this approach as an aid to protein purification has been demonstrated using human placental alkaline phosphatase. Following expression in either of two different eukaryotic expression systems, the CRD-phosphatase fusion protein can be isolated by one step of affinity chromatography on galactose-Sepharose under mild, non-denaturing conditions. Incorporation of a proteinase-sensitive linker allows cleavage of the CRD from the passenger protein. Immobilised proteinase could be rapidly separated from the cleavage products and the released, active phosphatase was purified away from the CRD by re-chromatography on galactose-Sepharose. These methods provide a means of isolating correctly folded recombinant eukaryotic proteins when cDNAs are available, but the properties of the encoded proteins are unknown.
Taylor
M E
ME
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032.
Drickamer
K
K
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015951:Factor Xa / Q000235:genetics* / Q000302:isolation & purification; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D016364:Reading Frames; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D015183:Restriction Mapping; D014357:Trypsin
D037263:Asialoglycoprotein Receptor; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D015951:Factor Xa