Martínez-Torrecuadrada Jorge Luis, Romero Silvia, Núñez Antonio, Alfonso Patricia, Sánchez-Céspedes Montserrat, Casal José Ignacio
Protein Technology Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncologicas (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
J Biotechnol. 2005 Jan 12;115(1):23-34. doi: 10.1016/j.jbiotec.2004.07.011.
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.
人LKB1，也称为STK11，是一种肿瘤抑制蛋白，在细胞增殖和极化中发挥重要功能。它可能是癌症治疗的一个重要靶点。为了生产大量重组蛋白用于生化和功能研究，一个全长cDNA克隆被亚克隆并在大肠杆菌和昆虫细胞中表达。尽管与带有6xHis、GST和MBP标签的LKB1对应的融合蛋白可以在大肠杆菌中过表达，但只有MBP-LKB1以可溶但严重降解的形式回收。进一步研究表明该蛋白无功能。随后在昆虫细胞中表达带有6xHis和GST标签的LKB1也产生了不溶性产物。然而，当伴侣蛋白Hsp70及其辅因子Hsp40和Hsdj与GST-LKB1共表达时，最终蛋白的溶解度明显增加。此外，这种可溶的、纯化的重组GST-LKB1被证明是一种磷蛋白，至少丝氨酸325残基被磷酸化。纯化的蛋白具有功能活性，因为它能够在没有任何相关激酶的情况下显示自磷酸化。
