An efficient expression system for the production of functionally active human LKB1.

Jorge Luis Martínez-Torrecuadrada; Silvia Romero; Antonio Núñez; Patricia Alfonso; Montserrat Sánchez-Céspedes; José Ignacio Casal

doi:10.1016/j.jbiotec.2004.07.011

PubMed · 15607222

An efficient expression system for the production of functionally active human LKB1.

Jorge Luis Martínez-Torrecuadrada·Silvia Romero·Antonio Núñez·Patricia Alfonso·Montserrat Sánchez-Céspedes·José Ignacio Casal

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Abstract

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.

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AMP-Activated Protein Kinase Kinases Baculoviridae Cloning, Molecular Enzyme Activation Escherichia coli Gene Expression Regulation, Bacterial Gene Expression Regulation, Enzymologic Genetic Vectors Humans Molecular Weight Protein Engineering Protein Serine-Threonine Kinases Recombinant Fusion Proteins Solubility Transfection Journal Article

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Jorge Luis Martínez-Torrecuadrada, Silvia Romero, Antonio Núñez, Patricia Alfonso, Montserrat Sánchez-Céspedes, José Ignacio Casal. 2005-01-12. An efficient expression system for the production of functionally active human LKB1.. https://doi.org/10.1016/j.jbiotec.2004.07.011

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