Expression and rapid purification of recombinant rat calretinin: similarity to native rat calretinin.
Rat calretinin coding region was subcloned into a prokaryotic expression vector (pGEX). The glutathione-S-transferase:calretinin fusion protein produced in Escherichia coli was purified on a glutathione-Sepharose affinity column. Recombinant rat calretinin was cleaved on the column by thrombin, eluted, and purified to homogeneity using DEAE-cellulose chromatography. Recombinant and native rat calretinin performed the same on DEAE columns, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), Western blots, and 45Ca overlay on nitrocellulose blots. The recombinant calretinin migrated similarly to the more basic (pI 5.3) of two forms of native calretinin demonstrated by two-dimensional SDS-PAGE. Calcium binding equilibria revealed identical apparent binding affinity and capacity. Difference(s) between native and recombinant did not affect the binding of calcium to calretinin or antibody recognition. Thus recombinant calretinin may be useful in the elucidation of possible cellular targets of native calretinin.
Strauss
K I
KI
NIMH, Laboratory of Clinical Science, Bethesda, Maryland 20892.
Kuźnicki
J
J
Winsky
L
L
Jacobowitz
D M
DM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
C576512:Calb2 protein, rat; D064032:Calbindin 2; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; D007544:Isopropyl Thiogalactoside; D002118:Calcium