Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli

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Abstract
A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257‐265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH‐HV) was isolated and subjected to trypsin digestion and RP‐HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C‐terminus (aa 179‐191) of hGH and contains one of the two disulfide bridges in hGH, viz. CySl82‐Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH‐HV (Tv18 + 19) and the corresponding tryptic peptide from rhGH (T18 + 19) were identical. Electrospray mass spectrometry (ES/MS) of Tv18 + 19 isolated from rhGH‐HV revealed a monoisotopic mass increase of 32.7, as compared to T18 + 19 from rhGH. A synthetic T18 + 19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragments in ES/MS compared to Tv18 + 19 isolated from rhGH‐HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cysl82‐SSS‐Cys189) bridge not found in the corresponding T18 + 19 disulfide peptide. Furthermore, the synthetic Tv18 + 19 co‐eluted in RP‐HPLC with T18 + 19 isolated from rhGH‐HV. Two‐dimensional NMR spectroscopy of the synthetic T18 + 19 and Tv18 + 19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (δ§>0.05 ppm) observed were for the β‐protons of Cys182 and Cys189 in Tv18 + 19 as compared to T18 + 19. CD spectroscopy data were also in agreement with the above results. Based on these physico‐chemical data, rhGH‐HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH‐HV was studied by a biosensor device, BIAcoreTM. The binding capacity of rhGH‐HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties. © Munksgaard 1996.