An efficient expression system for the production of functionally active human LKB1.
Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization. It might constitute an important target in cancer therapy. In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells. Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E. coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form. Further studies demonstrated that this protein was not functional. Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also. However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained. Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated. The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase.
Martínez-Torrecuadrada
Jorge Luis
JL
Protein Technology Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncologicas (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Romero
Silvia
S
Núñez
Antonio
A
Alfonso
Patricia
P
Sánchez-Céspedes
Montserrat
M
Casal
José Ignacio
JI
D000091162:AMP-Activated Protein Kinase Kinases; D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D014162:Transfection / Q000379:methods*
D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases