Abstract
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.
Highlights
From the $Howard Hughes Medical Institute and Departments of TPharmacology and 11Cell Biology, Vanderbilt University, Nashville, Tennessee 37232
Escherichia coliT. he genewas designed toallow rapid, conserved structurally. efficient changes of single or multiple amino acidsby While similar in primary structure, SlOOa and Sloop show using cassette-based mutageneswishile the geneis res- differences in their relative distribution among species and ident in the vector
The expressed prote(VinUSB-1)is tissues (Katoand Kimura, 1985;Zimmer and Van Eldik, indistinguishable from bovine braSilnO O f l in terms of 1987),and the patternsof S100a- and Sloop-binding proteins electrophoretic mobility,reactivity with antibodies to differ among tissues (Zimmer and Van Eldik, 1987)
Summary
S l O O Gene Expression and Purification of Expressed S l O O Protein 'The abbreviations used are: NEFn, eurite extension factor; (VUSB-1)-A transformant containing the SlOOgene insert was EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; IPTG, iso- cultured for 7 h in 200 ml of LB media (Gibco) containing 50 pg/ml propyl-8-D-thiogalactopyranoside;PCI, phenol/chloroform/isoamyl of ampicillin and thenplaced at 4 "C overnight. This culture was used alcohol; bp, base pairs; HPLC, high performance liquid chromatog- to inoculate 5 liters of LB media (20 ml of culture/liter of LB media)
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