Effective expression of recombinant cytotoxic protein via its attachment to a polyglutamine domain.
Inadvertent cytotoxicity may hinder the expression of many recombinant proteins that are of industrial or medicinal importance. Here, we show that covalent binding of the influenza A cytotoxic protein M2 to a polyglutamine domain (polyQ-M2; QM2) results in significant delay of its cytotoxic effects when compared to wild-type protein (M2wt). We also show that while expression of recombinant M2wt from A/WSN/1933 strain could not be attained in vaccinia virus (VV), polyQ-M2 was successfully expressed in this system. Moreover, we demonstrate that in cell culture, the polyQ domain is cleaved off following 48 h of expression, thus releasing free and active M2. Similarly, we show the spontaneous cleavage and polyQ release from fusion with another distinct polypeptide, green fluorescent protein (GFP). Expression of M2 from QM2 construct was more prolonged than one based on M2wt-expressing construct, markedly exceeding it at the later time points. Therefore, cell death caused by a toxic polypeptide may be suppressed via genetic fusion with polyQ, resulting in its enhanced expression, followed by slow release of the free polypeptide from the fusion. Collectively, covalent fusion with polyQ or other aggregate-forming domains presents a novel approach for industrial production of cytotoxic proteins and also holds promise for gene therapy applications.
Ilyinskii
Petr O
PO
Cure Lab, Inc., Canton, Massachusetts., USA.
Nurminskaya
Maria V
MV
Gabai
Vladimir L
VL
Prilipov
Alexei G
AG
Usachev
Evgeny V
EV
Zakharova
Lyudmila G
LG
Thoidis
Galini
G
Altstein
Anatoly D
AD
Shneider
Alexander M
AM
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
D049452:Green Fluorescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C097188:polyglutamine