Purification and molecular characterization of recombinant rat betacellulin.
A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy. Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC. Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa. Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography. Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources.
Dunbar
A J
AJ
Cooperative Research Centre for Tissue Growth and Repair, CSIRO Health Sciences and Nutrition, PO Box 10065 Adelaide BC, South Australia 5000, Australia. andrew.dunbar@gropep.com.au
Priebe
I K
IK
Sanderson
M P
MP
Goddard
C
C
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D066259:Betacellulin; D001665:Binding Sites; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D066246:ErbB Receptors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa; D006133:Growth Substances / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
C587851:BTC protein, human; D066259:Betacellulin; C587852:Btc protein, mouse; C587853:Btc protein, rat; D017931:DNA Primers; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D015951:Factor Xa