Bioactive recombinant neuregulin-1, -2, and -3 expressed in Escherichia coli.
The neuregulins (NRGs) are a family of signaling proteins that are ligands for receptor tyrosine kinase of the ErbB family (namely ErbB3 and ErbB4). To date, four different neuregulin genes have been identified (neuregulin1-4). While NRG1 isoforms have been extensively studied, little is yet known about the other genes of the family. We report the expression of recombinant NRG1beta1, NRG2alpha, NRG2beta, and NRG3 as recombinant fusion proteins in Escherichia coli. The cDNA encoding for the EGF-like domain of each protein was cloned from the mouse olfactory bulb and inserted into the pET-19b vector allowing for bacterial expression of the protein fused to an N-terminal His tag. The recombinant NRGs expressed in the inclusion bodies were solubilized under denaturing conditions, purified by affinity chromatography, and refolded via dialysis in the presence of reducing agents. Purified recombinant NRGs were active as they bound to their receptors and induced their phosphorylation. In particular, and in agreement with data on the native proteins, all the molecules were able to bind and activate ErbB4 while only the rNRG1 and the two rNRG2 (but not rNRG3) bound ErbB3.
Mautino
Beatrice
B
Department of Animal and Human Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.
Dalla Costa
Lorenza
L
Gambarotta
Giovanna
G
Perroteau
Isabelle
I
Fasolo
Aldo
A
Dati
Claudio
C
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020890:Neuregulin-1; D020880:Neuregulins; D018739:Oncogene Proteins v-erbB / Q000378:metabolism; D010766:Phosphorylation; D017510:Protein Folding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C486357:NRG3 protein, human; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020890:Neuregulin-1; D020880:Neuregulins; C490581:Nrg1 protein, mouse; C490580:Nrg1 protein, rat; C491453:Nrg2 protein, mouse; C491452:Nrg2 protein, rat; C108113:Nrg3 protein, mouse; D018739:Oncogene Proteins v-erbB; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins