Purification and characterization of the product of chemically synthesized human growth hormone gene expression in Escherichia coli.

Abstract

The efficient purification and characterization of the product of chemically synthesized human growth hormone (hGH) gene expressed in Escherichia coli are described. The product was purified from the cell lysates of the E. coli by means of ammonium sulfate precipitation, DE-52 chromatography, chromatofocusing chromatography and Ultrogel AcA 54 chromatography. The purified hGH gene product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, non-denaturing polyacrylamide gel electrophoresis, gel electrofocusing and high-performance liquid chromatography (HPLC). The purified product and an authentic methionyl hGH (m-hGH) showed identical behavior in these systems. The structural features of the purified product were examined by means of amino acid composition analysis, NH2-terminal sequence analysis and tryptic peptide mapping. The experimental values of the amino acid composition of the purified product were in agreement with the theoretical values for m-hGH. Its NH2-terminal sequence (39 amino acid residues) was identical with that of the published sequence of hGH, except for an additional amino-terminal methionine residue immediately preceding phenylalanine at residue 1. The elution profile of the tryptic peptides of the purified product on HPLC was identical with that of authentic m-hGH. These elution profiles were nearly identical with that of a pituitary-derived hGH, with the exception of one peak due to NH2-terminal peptide. On the basis of these results, the purified product was identified as m-hGH.