Assembly,Purification And Crystallization Of Eukaryotic Membrane Protein Complex CD147-MCT1

Monocarboxylate transporter 1(MCT1)is a multiple transmembrane protein belonging to the superfamily of solute carrier transporters,which can mediate the transmembrane transport of lactic acid produced by glycolysis,regulate the energy metabolism of tumor cells and maintain the balance of tumor acidic microenvironment.CD147 is a type I integrated transmembrane glycoprotein belonging to the immunoglobulin superfamily.It is highly expressed in a variety of malignant tumors and can mediate invasion,metastasis,proliferation and adhesion of tumor cells as well as tumor angiogenesis.CD147,as a chaperone protein,binds to MCT1 to facilitate the carrier to localize into the plasma membrane correctly and regulate its expression level and transport activity on the plasma membrane.In order to elucidate the structural and functional relationship of the CD147-MCT1 protein complex and the molecular mechanism of its activity regulation during cancer development,we carried out a three-dimensional structure study of the CD147-MCT1 protein complex and its binding substrate or inhibitor complex.The analysis of the structure of the complex will also lay an important structural foundation for the development of anti-tumor targeted drugs.In this paper,the CD147-MCT1 protein complex was used as the research object,and the complex was successfully expressed and assembled using the baculovirus-insect cell system.Through systematic optimization of recombinant expression and purification conditions,stable expression of the CD147-MCT1 protein complex was obtained,and the protein expression of the complex was ～3 mg/L.Preliminary biochemical analysis shows that the protein complex exists as a heterodimer.The results of dynamic light scattering analysis show that the combination of substrate L-lactic acid is conducive to the stability of the conformation of the complex,making the sample aggregation state and conformation more uniform.Through crystallization screening and optimization,crystals of the composite sample of CD147-MCT1 binding substrate L-lactic acid were obtained.Further crystallization optimization can obtain a single crystal,but its diffraction resolution is lower than 14 A,which shows that the crystal has serious anisotropy problems,and the crystallization conditions need to be further optimized.In addition,we also obtained crystals of CD147 full-length protein and collected the entire set of diffraction data with a resolution of about 3.4 A.Structural analysis is in progress.In order to optimize the crystal quality and improve the diffraction resolution,we conducted a systematic restriction protease degradation analysis of the CD147-MCT1 protein complex sample,combined with the secondary structure prediction analysis,to find the flexible loop region of the target protein.And based on this,CD147 and MCT1 proteins were modified by truncating the flexible region,in order to obtain high-quality samples conducive to the orderly accumulation of crystals and improve the quality of crystal diffraction.At present,4 kinds of flexible region truncated protein complexes with stable expression and uniform state have been obtained,and the subsequent crystal optimization is still in progress.