Accurate processing and secretion in the baculovirus expression system of an erythroid-cell-stimulating factor consisting of a chimaera of insulin-like growth factor II and an insect insulin-like peptide.
A synthetic gene encoding the signal peptide and the N-terminal sequence of bombyxin, an insect insulin-like peptide, and the 58 amino acids of the C-terminal sequence of human insulin-like growth factor II (IGF II) has been expressed using the baculovirus system. This synthetic chimaera was obtained by amplification of four overlapping oligonucleotides using Taq polymerase and cloning into the transfer vector pBluebac. The construct was integrated by homologous recombination into the Autographa californica nuclear polyhedrosis genome. Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus secreted an accurately processed peptide consisting of the ten N-terminal amino acids of bombyxin and the 58 C-terminal amino acids of IGF II. The N-terminal glutamine of bombyxin was changed to asparagine to facilitate sequencing of the synthetic peptide. The chimaera was five times more potent than human recombinant IGF II in its capacity to stimulate thymidine incorporation into erythroid cells of fetal bovine liver in a serum-free medium. It stimulated erythroid colony formation in the presence of 2 microunits/ml erythropoietin in cells cultured over a monolayer of stromal cells of fetal liver. Artificial chimaeras as described here may prove useful for the production of insulin, IGF I and other peptides as secreted proteins in insect cells.
Congote
L F
LF
Department of Medicine, McGill University, Montreal, Canada.
Li
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D004920:Erythropoiesis / Q000187:drug effects; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009036:Moths; D009479:Neuropeptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism
D004274:DNA, Recombinant; D009479:Neuropeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C075028:bombyxins; D007335:Insulin-Like Growth Factor II; D013936:Thymidine