Production of biologically active N alpha-desacetylthymosin alpha 1 in Escherichia coli through expression of a chemically synthesized gene.
Thymosin alpha 1, an immune restorative polypeptide hormone, was synthesized in Escherichia coli by using recombinant DNA cloning techniques. Based on the known amino acid sequence, a gene coding for the thymosin alpha 1 polypeptide chain was designed and enzymatically assembled from chemically synthesized oligodeoxyribonucleotide fragments. The gene was ligated into plasmid pBR322 and placed under lac operon control, and N alpha-desacetylthymosin alpha 1 was expressed as part of a beta-galactosidase chimeric protein. Cyanogen bromide cleavage of this protein gave a mixture of polypeptides, among which thymosin alpha 1 activity was detected by radioimmunoassay (RIA). The E. coli product is identical with native thymosin alpha 1 isolated from calf thymus in the amino acid sequence but lacks the N-terminal acetyl group. Results of a guinea pig migration inhibition factor (MIF) assay, a terminal deoxyribonucleotidyl transferase (TdT) assay, and radioimmunoassay indicate that the N alpha-desacetylthymosin alpha 1 produced by deoxyribonucleic acid (DNA) cloning techniques has biological activity equivalent to that of the native hormone.
D000595:Amino Acid Sequence; D000818:Animals; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011863:Radioimmunoassay; D013947:Thymosin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013951:Thymus Hormones / Q000096:biosynthesis*
D008263:Macrophage Migration-Inhibitory Factors; D013951:Thymus Hormones; D013947:Thymosin; C026456:desacetylthymosine alpha(1); D004253:DNA Nucleotidylexotransferase