Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry.
We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.
Jung
Chulho
C
LG Life Sciences Ltd/R&D Park, 104-1 Munji-dong, Yuseong-gu, Daejeon 305-380, South Korea.
Lee
Young-Phil
YP
Jeong
Yeong Ran
YR
Kim
Jin Young
JY
Kim
Young Hwan
YH
Kim
Hyun Sik
HS
D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019382:Human Growth Hormone / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D011994:Recombinant Proteins; D019382:Human Growth Hormone; C447442:somatrem