The structural properties of purified human growth hormone (hGH) produced by Escherichia coli K-12 into which the hGH gene has been inserted have been fully characterized by high-pressure liquid chromatography of native hGH and tryptic digests of hGH. All of the tryptic peptides have been separated by high-pressure liquid chromatography and their sequence determined. Comparison of the primary structure with that of the purified pituitaryderived hGH has established the integrity of the biosynthetic hGH disulfide arrangement and amino acid sequence with the presence of an extra NH?-terminal methionine. ' An abstract of these results was presented at the International Symposium on High-Pressure Liquid Chromatography of Proteins and Peptides, held in Washington, D. C., on November 16-17, 1981. r Abbreviations used: hGH, human growth hormone; HPLC, high-pressure liquid chromatography; PTH, phenylthiohydantoin; TFA, trifluoroacetic acid. tensive sequence data of these two proteins. By employing a high-pressure liquid chromatography (HPLC) peptide-mapping procedure coupled with microsequencing techniques we have established the integrity of the biosynthetic hGH amino acid sequence and disulfide arrangement using less than 50 nmol of starting material. EXPERIMENTAL PROCEDURES Reagents. Biosynthetic hGH was purified from E. coli K-12 extracts as described previously (2). Purified pituitary-derived hGH was provided by Kabi AB, Stockholm, Sweden. Trypsin which had been treated with L-[ tosylamido-Zphenyl] ethyl chloromethyl ketone was purchased from Worthington. Dithiothreitol was from Sigma Chemical Company and prepacked, disposable PD-10 columns containing Sephadex G-25 were purchased from Pharmacia Fine Chemicals. Constant boiling hydrochloric acid, trifluoroacetic acid (Sequanol grade), and Polybrene were obtained from Pierce Chemical Company. HPLC-grade acetonitrile, n-propanol, and methanol were obtained from Burdick and Jackson Chemical Company. Ethyl acetate and benzene (Resi-analyzed)