Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells.
The 16-kDa fragment of prolactin (16-kDa PRL), derived from proteolytic cleavage of 23-kDa PRL, was shown to have antiangiogenic activity. Previous studies have shown that recombinant 16-kDa PRL produced from bacteria often contained endotoxins, which are cytotoxic to endothelial cells, and varied in its biological activity due to changes in its refolding from inclusion bodies. These problems limited the use of recombinant 16-kDa PRL. To improve the generation of recombinant 16-kDa PRL, we expressed 16-kDa PRL in Sf9 insect cells using a baculoviral expression system. The signal sequence of the human PRL gene and codons for seven histidines were added to the N- and C-termini, respectively, of the 16-kDa PRL cDNA construct. Recombinant 16-kDa PRL was detected in both the cell pellet and the medium. About 0.28 mg purified protein was isolated from the cell pellet of 4 x 10(7) infected cells using nickel affinity chromatography. Sixteen kilodalton PRL was posttranslationally modified with apparent molecular weights of 16 and 18 kDa on SDS-PAGE. The level of 18-kDa protein was significantly reduced after digestion with peptidyl-N-glycosidase, suggesting that the heterogeneity was due to glycosylation of 16-kDa PRL. N-terminal sequence analysis confirmed the fact that both proteins were human 16-kDa PRL and the signal sequences were cleaved at the same position as that of human PRL. Consistent with its role as an angiogenesis inhibitor, purified recombinant 16-kDa PRL inhibits the proliferation of endothelial cells with a potency similar to that previously reported for the protein generated in Escherichia coli. This 16-kDa PRL expressed in Sf9 cells is a useful reagent for functional studies and for the purification and identification of its receptor.
Galfione
Matthew
M
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
Luo
Weiping
W
Kim
Jeri
J
Hawke
David
D
Kobayashi
Ryuji
R
Clapp
Carmen
C
Yu-Lee
Li-Yuan
LY
Lin
Sue-Hwa
SH
D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification*; D011388:Prolactin / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D018411:Spodoptera
D020533:Angiogenesis Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011388:Prolactin