Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis.
As an initial step in studies aimed at addressing the question of what common and unique features of the S100 family of proteins are related to their specific functions and localizations, a gene coding for one of the S100 proteins, S100 beta, has been prepared by ligation of 12 overlapping, synthetic oligonucleotides. Automated DNA sequence analysis demonstrated that the final construct has the expected structure. The gene was inserted into a plasmid vector that contains a tac promoter and ampicillin-resistance gene, thus allowing both amplification and direct expression cloning in Escherichia coli. The gene was designed to allow rapid, efficient changes of single or multiple amino acids by using cassette-based mutagenesis while the gene is resident in the vector. The expressed protein (VUSB-1) is indistinguishable from bovine brain S100 beta in terms of electrophoretic mobility, reactivity with antibodies to S100 beta, amino acid composition, and partial amino acid sequence analysis. Preparations of expressed protein are also functionally similar to bovine brain S100 beta as determined by aldolase activator activity and neurite extension factor activity, supporting the concept that these activities are a property of the S100 beta polypeptide.
Van Eldik
L J
LJ
Howard Hughes Medical Institute, Vanderbilt University, Nashville, Tennessee 37232.
Staecker
J L
JL
Winningham-Major
F
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D009414:Nerve Growth Factors; D010449:Peptide Mapping; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000235:genetics*
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins