Expresssion And Purification Of Auxin Binding Proteins And Phosphate Sensing Proteins Complex

Part 1.Expression and Purification of Auxin Binding Proteins.Today,studies are now focusing on the high efficiency of gene expression.Although E.coli expression system is easy to use and cultivate.However,many proteins cannot be expressed in bacteria.So,we are trying to develop a Eukaryotic expression system and hope to promote this system to biology,medicine,agriculture and other fields.Auxin is an important phytohormone for plant growth and develpment.Auxin binding proteins may play a vital role in auxin perception and signaling.This research expressed and purified auxin binding proteins using different expression systems.Two expression systems were tried to express the auxin binding proteins,the Escherichia coli prokaryotic expression system and the insect cell baculovirus expression system.In the prokaryotic system,auxin binding proteins were expressed but insoluble.Adding an Msyb tag may promote the solubility of these proteins while size exclusion chromatography showed that these proteins are highly aggregated.In the insect cell system,auxin binding proteins were well expressed and soluble.Size exclusion chromatography showed ABP1 were not aggregated.We also successfully expressed and secreted Auxin binding protein 1(ABP1)and Transmembrane Kinase 1(TMK1)extracellular domain from High5 cells through secretion pathway.However,no strong interaction was detected between ABP1 and EXTMK1 through size exclusion chromatography under pH 5.7 and high concentrations of 1-naphthylacetic acid(NAA).This may indicate that the interaction between ABP1 and EXTMK1 could be weaker or other protein factors may join in to form a complex.This study provides significant protein properties of auxin binding proteins through detailed expression and purification methods and promotes further research on the mechanism of auxin signaling pathway.This study also provides a novel insect cell baculovirus system for the expression of eukarytic proteins.Part 2.Purification and Crystallization of Phosphate Sensing Proteins Complex.It is now much more convenient to use Bacteria as an expression host to produce ideal proteins.However,many proteins may form inclusion body in bacterias and protein complexes are much more difficult to express in bacterias.This study aimed to find proper method to produce soluble proteins complex in E.coli.Inorganic phosphate is one of the essential nutrient element for all the organisms.The core proteins involved in phosphate sensing are PH081?PHO80-PHO85 three-protein complex.We firstly tried the expression of PH081 full length and several truncations in bacteria.PH081 full length proteins were soluble however the expression level was low.Different truncations of PH081 formed inclusion bodies in bacterias.However,only PH081-MD domain fused with an Msyb tag were well expressed and purified.This study also purified the PHO80-PHO85 two proteins complex by using co-expression system.The crystals of PHO80-PHO85 complex were formed by screening of more than 2000 conditions.Size exclusion chromatography showed a strong interaction between PHO80-PHO85 and PH081-MD.This study successfully assembled the three-protein complex PH081?MD-PHO80-PHO85 and detected their interaction.Purification and crystallization of PHO proteins provide valuable experience and background for further research into the PHO pathway related proteins.This research developed a co-expression cloning method and may promote the co-expression and purification of multi-proteins complex in protein engineering.