STUDIES ON THE MICROHETEROGENEITY AND IN-VITRO ACTIVITY OF GLYCOSYLATED AND NONGLYCOSYLATED RECOMBINANT HUMAN PROLACTIN SEPARATED USING A NOVEL PURIFICATION PROCESS

Recombinant human PRL was produced in a murine C127 cell expression sy stem and purified to greater than 97% homogeneity using anion and cati on exchange chromatography. This material was biologically equivalent to pituitary-derived PRL in both an enzyme-linked immunosorbent assay and the Nb-2 lymphoma cell proliferation assay. The predominant PRL fo rms were identified by sodium dodecyl sulfate-polyacrylamide gel elect rophoresis and immunoblotting as being 23 and 25 kilodaltons (kDa). Th ese mass values were confirmed by electrospray mass spectroscopy. Glyc osidase digestions indicated that the 25-kDa PRL is N-glycosylated and sialylated, whereas 23-kDa PRL is nonglycosylated. Glycosylated and n onglycosylated forms of the hormone were individually purified to grea ter that 95% homogeneity using novel cation exchange chromatography. I soelectric focusing demonstrated that both forms consist of multiple c harge isomers, with the charge heterogeneity of the glycosylated form primarily due to differences in sialylation. Monosaccharide analysis o f the glycosylated form suggested a minimal complex oligosaccharide ch ain that may be fucosylated and partially sialylated. Oligosaccharide mol mt were determined by electrospray ionization mass spectroscopy. A nalysis of the oligosaccharides by fluorophore-assisted carbohydrate e lectrophoresis indicated that bi- and triantennary oligosaccharide for ms are predominant and have multiple combinations of terminal sialylat ion. Both forms of PRL were active in the Nb-2 lymphoma cell prolifera tion assay; however, the 23-kDa nonglycosylated form was 3-4 times mor e active in this assay than the 25-kDa glycosylated form.