Overexpression And Purification Of Human Ferroportin

The solute carriers family is the second largest family of membrane proteins in the human genome and the largest family of membrane transporters.It is widely found in prokaryotes and eukaryotes.SLC family proteins are located on the cell membrane and transport a variety of solutes.They are responsible for regulating the intake and output of various essential substances,such as amino acids,inorganic ions,neurotransmitters,hormones,vitamins and drugs,etc,and play important roles in a variety of cells.The purpose of this paper is to select proteins with stable properties and high expression from SLC proteins with important physiological significance and potential as drug development targets for large expression and purification for subsequent crystallization or electron microscopy.In this project,preliminary screening was first performed on many SLC family proteins with important physiological significance,and ten proteins were found to have high expression level and stable structural properties.This method does not require large amounts of protein expression and purification,and only nanogramlevel unpurified proteins can be detected.One of transporters SLC40A1(FPN1)was selected for preliminary structural investigation.SLC40A1(FPN1)is an iron transporter,which is currently the only known protein that regulates iron export.SLC40A1 participates in the iron efflux mechanism,and is critical in regulating iron balance inside and outside the cell.Mutations in SLC40A1 gene are related with hereditary diseases.Studying the structural properties of SLC40A1 would be important for understanding the mechanism of iron transfer and related diseases.The second part of this subject mainly focused on expression and purification of SLC40A1.We optimized the expression by using different expression systems,the expression system was optimized for expression in mammalian cells and insect cells,mammalian cells were cultured in two ways: suspension and adherence,and it was found that SLC40A1 had the highest expression level and stable structural properties in insect cells,and subsequently expressed a large amount in insect cells.And purified the SLC40A1 protein by multiple steps including detergent solubilization,affinity chromatography,MBP tag removal and size exclusive chromatography,SLC40A1 protein was obtained with good purity,homogeneity,and stability.Lastly LCP crystallization was carried out to look for suitable conditions to crystallize SLC40A1.These works paved the way for the structural studies and elucidation of the transport mechanism of SLC40A1.