Affinity purification and microcharacterization of recombinant DNA-derived human growth hormone isolated from an in vivo model.
A procedure has been developed for the isolation and purification of trace amounts of unlabeled proteins from biological solutions. Using a combination of affinity chromatography and reversed-phase HPLC, microgram amounts of recombinant DNA-derived human growth hormone (rhGH) were purified from an in vivo rat model. Microcharacterization techniques were developed, and picomole amounts of the recovered protein were digested with trypsin and characterized using capillary HPLC peptide mapping. The described procedures were used to study the chemical changes that occur in rhGH following intravenous administration. The study demonstrated that both deamidation and oxidation can occur in vivo, although the former would occur to a significant extent only in proteins with an extended half-life.
Genentech, Inc., South San Francisco, California 94080.
J E
JE
Battersby
V R
VR
Mukku
R G
RG
Clark
W S
WS
Hancock
D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004274:DNA, Recombinant; D013006:Growth Hormone / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D010449:Peptide Mapping; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D013006:Growth Hormone; D014357:Trypsin