The Methodology Development Of Protein Labeling And Protein Delivery

Protein is the material foundation and main executor of life activity.Protein labeling and delivery are importanttechniques for studying the protein's localization,transport,biological function and so on.Therefore,it is very important to develop efficient,facile protein labeling and delivery methods.In this dissertation,we developed new protein labeling and protein delivery methods.These methods process the potential to be applied for the research field of protein and drug development.Protein labeling method is a common method to study the distribution,structure and function of protein in cells.The FRET is the most important technology for studying the structure and function of proteins.To use FRET technology,the target protein need to be dual-labeled.Therefore,it is very important to develop an efficient and convenient dual-labeling method.We have developed a novel and efficient dual-labeling method using SUMO protein expression system and intein technology.Using this method,we have prepared a dual-labeling ubiquitin protein.And it was applied to the screening of the inhibitors of UCH-L3.The prepared fluorescent dual-labeling ubiquitin protein was proved to be a sensitive and efficient UCH-L3 sensor.Meanwhile,we developed an efficient protein delivery method based on our developed protein labeling method.Adjacent benzene boric acid formaldehyde can react with cysteine and cysteine derivatives less than 10 min and high efficiency without adding any other agent.And the pH of this reaction can be 3 to 9.The ubiquitin protein was labeled successfully by the reaction of the N terminal ubiquitin protein with the adjacent benzene boric acid formaldehyde.This method has been proved to be a very efficient and convenient method for protein labeling.Using positively charged liposomes,we developed an efficient liposome mediated protein delivery method.The experiments show that this protein delivery method can efficiently transport nM target protein into the cell.Meanwhile,we have developed an efficient receptor mediated protein delivery method based on our developed protein labeling method.The target protein can be transported to the cell by the receptor mediated way.By adding different amount of ortho phenyl borate and its analogues to the cell,we can regulate the transported protein to the intracellular cell.In summary,we have developed efficient,novel protein labeling and protein delivery methods.And this method can be applied to the research field of protein and drug development.