Characterization of the microheterogeneity of recombinant primate prolactin: implications for posttranslational modifications of the hormone in vivo.
Recombinant baboon and monkey prolactins were expressed in murine C127 cells. The hormones were purified from the conditioned media of these cells using a combination of cation, anion, and gel filtration chromatographies. This purification scheme provided approximately a 20-fold purification of the proteins with a 40% cumulative yield. Sodium dodecyl sulfate gel electrophoresis of the purified hormones in conjunction with Coomassie blue staining and immunoblotting procedures revealed three major prolactin-related bands with molecular weights corresponding to Mr 16,000, 23,000, and 27,000. Based on these analyses the samples were judged to be greater than 90% pure. Amino terminal sequence analysis of the purified baboon and monkey hormones provided three distinct prolactin-related sequences for each preparation. The predominant sequence corresponded to the predicted amino terminal sequences of the hormones which began with leucine at position 1. Two minor sequences, individually representing approximately 10-20% of the total population, were also identified; one starting at position 11 and the other at position 133. Carbohydrate compositional analysis of the proteins suggested that greater than 50% of the population were glycosylated with a fucosylated complex oligosaccharide. Analysis of the specific bioactivity of the recombinant hormones in the Nb2 cell proliferation assay showed them to be comparable to the NIH and WHO human pituitary-derived standards.
Recombinant Protein Development Department, Genzyme Corporation, Framingham, Massachusetts 01701.
E S
ES
Cole
E H
EH
Nichols
K
K
Lauziere
T
T
Edmunds
J M
JM
McPherson
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D010215:Papio; D011388:Prolactin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D011388:Prolactin