Dual Recognition Group Of Molecularly Imprinted Polymers Enriched Natural Trace Protein

This dissertation is composed of two parts: the first part investigated separation/enrichment of active natural low content protein using protein imprinted polymer; The second part of this dissertation studied de novo cloning of FKBP23 cDNA from pig ER using nested PCR and protein expression.Protein imprinted polymer studied in the first part is based on previous research production of our laboratory. In addition to small molecules, proteins can also be used as templates. There are thousands of types of protein within a cell and most are present at relatively low levels. Indeed, some proteins can exist in just a few copies, even though they perform important biological functions within a cell. Molecular-cloning techniques allow scientists to study these proteins by producing them in large quantities from cloned genes. However, cloned proteins are not identical to natural ones in vivo. Therefore, researchers are interested in purifying natural proteins from cell extracts all the time. In previous work, we reported a new method for protein imprinting with the assistant recognition polymer chains (ARPCs), which shows good recognition specificity.In this work, we synthesized a new type of the assistant recogniton polymer chains with random distributed double recognition sites. Pyridyl and carboxyl groups were used as recognition sites. Cloned pig cyclophilin 18 (pCyP18) was used as template. The template protein was selectively assembled with ARPCs from their library. These assemblies of protein and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, the synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 200 times. The concentration of natural pCyP18 adsorbed in eluent in this work enables the assay of PPIase activity to be performed. The assay of peptidyl-prolyl cis-trans-isomerase (PPIase) activity showed that natural pCyP18 is more active than cloned pCyP18 and, in particular, it is much more sensitive to the suppressant cyclosporine A (CsA). The second part of this thesis studied de novo cloning of FKBP23 cDNA from pig ER using nested PCR and protein expression. We hope to improve the eurytopicity of protein imprinted polymer and research natural protein. FKBP23 is a glycoprotein retained in endoplasmic reticulum (ER). If cloned pig FKBP23 as template protein can be prepared, natural pig FKBP23 in the ER can be acquired by protein imprinted polymer. We are interested in purifying natural FKBP23 and researching the function of glycosyl in FKBP23. Pig liver is a handy and abundant source to acquire the ER.The sequence of pFKBP23 was not reported. According to two highly conservative regions of nucleotide sequences of several detected species, we designed primers and used PCR to amplify the middle segment of the pFKBP23 gene. From this sequence we used nested PCR to extend the nucleotide sequence towards 5' and 3' ends for detecting the full length cDNA encoding pig FKBP23. Our result is available from GenBank with accession No. EU545235. We constructed a plasmid to express pFKBP23. Furthermore, we proved that the recombinant pFKBP23 can specifically bind to natural BiP, and the binding is interrelated with the Ca²⁺concentration. These will be a foundation to research natural pFKBP23.