Phosphoric acid entrapment leads to apparent protein heterogeneity.
Recombinant proteins produced in prokaryotes or eukaryotes show certain types of heterogeneity due to post-translational modifications. Some preparations of a soluble interferon gamma receptor, produced in Escherichia coli, appeared as a double band with slightly different mobilities in non-reducing sodium dodecylsulfate and native polyacrylamide gels. Ion spray mass spectrometry showed that the two forms had a mass difference of one to three multiples of 97 +/- 2 D. Gas chromatography-mass spectrometry analysis revealed the presence of phosphoric acid in the hydrolysate and in the intact protein. The more slowly migrating protein species had trapped molecules of phosphoric acid during the protein extraction. Most of the trapped phosphoric acid was loosely associated with the protein. One to three molecules were tightly, but non-covalently linked per receptor molecule. Phosphoric acid entrapment did not affect biological activity and most likely did not affect protein conformation. The species carrying phosphoric acid showed higher solubility. Trapping of phosphoric acid by proteins may be a general phenomenon and the results reported here thus useful in the characterization of other recombinant proteins.
Fountoulakis
M
M
F. Hoffmann-La Roche Ltd., Department of Gene Technologies, Basel, Switzerland.
Vilbois
F
F
Oesterhelt
G
G
Vetter
W
W
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D006358:Hot Temperature; D006801:Humans; D013058:Mass Spectrometry; D010756:Phosphoric Acids / Q000032:analysis*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D014508:Urea; D000096743:Interferon gamma Receptor
D010756:Phosphoric Acids; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D014508:Urea; D000096743:Interferon gamma Receptor; C030242:phosphoric acid