Quantifying biosynthetic human growth hormone in Escherichia coli with capillary electrophoresis under hydrophobic conditions.
A method has been developed which is able to quantitate the content of precursor biosynthetic human growth hormone (Pre-bhGH) in the cytosol of E. coli cells containing the gene for human growth hormone (hGH). The method uses hydrophobic C18 coated capillaries with native biosynthetic human growth hormone (bhGH) as an internal standard. This allows for highly robust and precise determinations as well as the evaluation of the presence of deamidated forms in the cytosol samples. Furthermore, by modifying the running buffer with zwitterionic surfactants and an organic modifier, it is possible to detect a related form with a three sulfur atom Cys-Cys bridge (trisulfide Pre-bhGH). Thus, a strong tool for monitoring the effect of fermentation conditions on the biosynthesis of bhGH is obtained.
Jørgensen
T K
TK
Health Care, Novo Nordisk A/S, Gentofte, Denmark.
Bagger
L H
LH
Christiansen
J
J
Johnsen
G H
GH
Faarbaek
J R
JR
Jørgensen
L
L
Welinder
B S
BS
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D019382:Human Growth Hormone / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D012015:Reference Standards; D015203:Reproducibility of Results
D011498:Protein Precursors; D011994:Recombinant Proteins; D019382:Human Growth Hormone