Expression of bovine intestinal calcium binding protein from a synthetic gene in Escherichia coli and characterization of the product.
Intestinal calcium binding proteins (ICaBP's) constitute a group of small vitamin D inducible proteins considered to play an important role in the absorption of dietary calcium. The mammalian ICaBP's are representatives of the "EF-hand" family of calcium binding proteins. As a first step in the application of protein engineering techniques to the study of structure-function relationships in mammalian ICaBP's, we have synthesized a gene encoding the minor A form (the native form lacking the two N-terminal amino acids) of bovine ICaBP employing a rapid, microscale gene synthesis technique based on "shotgun ligation" of sets of oligonucleotides. Expression of the synthetic gene from a plasmid containing the tac promoter in a lon protease deficient strain of Escherichia coli yielded the desired product at a level of about 1-2 wt % of total protein. During the purification of the ICaBP expressed in E. coli, a contaminant was strongly adhering to it but was efficiently removed by gel filtration after denaturation with urea. The minor A form of ICaBP produced in E. coli was characterized by its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by its total amino acid composition, partial amino acid sequence, UV absorption spectrum, and 360-MHz 1H NMR spectrum, showing beyond reasonable doubt its identity with the minor A form of ICaBP obtained from bovine intestines.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005813:Genes, Synthetic; D007413:Intestinal Mucosa / Q000378:metabolism*; D010957:Plasmids; D064030:S100 Calcium Binding Protein G / Q000235:genetics*; D014158:Transcription, Genetic
D064030:S100 Calcium Binding Protein G; D004262:DNA Restriction Enzymes