CHO-recombinant human growth hormone as a protease sensitive reporter protein.
Protein-free media are gaining more and more interest in mammalian cell culture technology. However, the range of commercially available protein-free media is wide, but lack of serum causes the lack of various substances (Keenan et al. in Cytotechnology, 50(1-3):49-56, 2006) which must be substituted case by case. Details on the composition of protein-free media are often unavailable or inaccessible in some cases, and as a consequence, there is an obvious need for testing procedures in order to evaluate the various commercialised products for their performance. Additionally, negative effects of tryptic meat digests on product quality have been reported in the literature (Gu et al. in Biotech Bioeng 56 (4):353-341, 1997). In the present studies of comparing various protein-free media for their suitability in propagation of recombinant CHO cells expressing human growth hormone (hGH), we have found somatotropin to be an excellent candidate for detection of protease activity. Somatotropin contains protease recognition sites for numerous proteases located around amino-acid residues 134-150. In this study, we demonstrate highly specific cleavage of recombinant hGH during batch cultivation. Analysis of the digested molecule was then performed by convergent methods like SDS-PAGE, HPLC and mass spectroscopy, and the results indicate hGH to be an ideal candidate for media and component screening in mammalian cell culture.
Kunert
Renate
R
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190, Vienna, Austria. renate.kunert@boku.ac.at
Steinfellner
Willibald
W
Altmann
Friedrich
F
Wallner
Jakob
J
Katinger
Hermann
H
Vorauer-Uhl
Karola
K
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism*
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D010447:Peptide Hydrolases