Utilizing The Protein Imprinted Polymer To Study The Interaction Of Proteins And N-terminal Sequence Of Natural BiP

This thesis is divided into two parts:preparation of the protein-imprinted polymer (PIP) with pyridine identification groups and protein-imprinted polymer as antibody substitute to study the interaction between BiP and FKBP23are discussed in the first part, and a two-step method for the separation and purification of natural low content and high molecular weight protein BiP and N-terminal sequence of BiP are investigated in the second part.Immunoprecipitation is an important method to study protein-protein interactions. The polyclonal or monoclonal antibody for target protein needs to be prepared which is a complicated process and the target protein, up to a certain concentration, can bind with antibody to form the precipitate, thus this method only can study the proteins with high expression amount and has limitations on the application.In the first part, we synthesized assistant recognition polymer chains (ARPCs) containing the randomly distributed pyridine identification groups and double bond anchor groups an assistant functional monomer. We used double-bond-functionalized macroporous resin as solid support, the cloned proteins mouse BiP (mBiP) and pig FKBP23(pFKBP23) as a template respectively to synthesize two kinds of protein-imprinted polymers of mPIPBiP and mPIPFKBP23which were used to adsorb the endoplasmic reticulum extract from pig liver. The obtained elutions were detected by silver staining and western-blot. BiP and FKBP23both existed in the eluents of mPIPBiP and mPIPFKBP23which showed that BiP can bind specifically to FKBP23in endoplasmic reticulum. When adjusting the concentration of Ca2+in buffer to4mmol, we detected BiP but could not detect’FKBP23from the eluent of mPIPBiP. Simultaneously, we detected FKBP23but could not detect BiP from the eluent of mPIPFKBP23which showed that BiP did not bind to FKBP23at4mmol [Ca2+] and the binding between BiP and FKBP23was interrelated with the concentration of Ca2+. These results were in accordance with that obtained by co-immunoprecipitation method using antibody. So we can confirm that protein-imprinted polymer can be used as antibody substitute to study the protein-protein interaction.BiP is one of the endoplasmic reticulum Hsp70family members, playing a significant role in the endoplasmic reticulum secretory proteins and membrane protein folding, storage and transfer. The second part of the work is to purify the pig BiP protein using two kinds of imprinted polymers prepared with the cloned mBiP as a template from the Pig endoplasmic reticulum extract. By contrast, we found that two kinds of protein-imprinted polymers for the non-specific protein adsorption was different. Therefore, we will isolate and purify pig BiP through the continuous use of two kinds of protein-imprinted polymers to achieve the purpose of purification BiP. At this stage, we did not get the result of the N-terminal sequence of purified BiP, and the reason leading to this result may be the N-terminus of natural BiP closed or closed during the extraction process. In the future, we will continue to research the N-terminal sequence of the BiP through the combination of mass spectrometry and Edman degradation.