High-level expression of functional tumor suppressor LKB1 in Escherichia coli.
The human LKB1 tumor suppressor has been implicated as an important regulator of many cellular processes and signaling pathways, indicating that it could be a good candidate for anticancer drugs. The failure of its obtain high-level expression has been a major obstacle to study its protein structure and function in vitro. Here, we describe the high-level expression of human LKB1 in Escherichia coli and show its kinase activity and anticancer effects on a tumor cell line. The gene encoding LKB1 was optimized by replacing rare codons with codons frequently used in E. coli and synthesized with overlapping primers. The recombinant His-LKB1 was expressed in hosts BL21(DE3) (BL) and Rosetta-gami(DE3)pLysS (RG). His-LKB1 from BL was present mainly as inclusion body. The soluble His-LKB1 from RG accounted for 34.1% of total proteins and the yield of purified His-LKB1 was approximately 92 microg/ml. Purified His-LKB1 protein from both hosts was functionally active, as shown by reversible autophosphorylation and kinase activity in the absence of any other associated kinase. The growth inhibitory ratio of the purified BL-derived and RG-derived His-LKB1 on hepatic carcinoma SMMC-7721 cells was 24.97% and 45.68%, respectively, and both could produce significant cell-cycle arrest.
Liu
Jun'e
J
College of Life Sciences, Inner Mongolia University, Hohhot 010021, China.
Hu
Tingmao
T
Hou
Xin
X
D000091162:AMP-Activated Protein Kinase Kinases; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D004926:Escherichia coli; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology
D006131:Growth Inhibitors; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases