Affinity regulation of proteins with thermoresponsive polymer-brush interfaces

2007 Fiscal Year Final Research Report Summary

• Project/Area Number: 18500367
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biomedical engineering/Biological material science
• Research Institution: Tokyo University of Science
• Principal Investigator: KIKUCHI Akihiko Tokyo University of Science, DEPT MATER SCI & TECHNOL, ASSOCIATE PROFESSOR (40266820)
• Project Period (FY): 2006 – 2007
• Keywords: THERMORESPONSIVE BRUSH INTERFACES / POLY (N-ISOPROPYLACRYLAMIDE) / IMMOBILIZED METAL ION AFFINITY / ATOM TRANSFER RADICAL POLYMERIZATION / PROTEIN / IMINODIACETIC ACID / NITRILOTRIACETIC ACID

Research Abstract

In this research, we aimed to prepare novel thermoresponsive interfaces modulating immobilized metal ion affinity regulation of bioactive proteins. To realize this aim, we prepared thermoresponsive polymer-grafted surfaces with iminodiacetic acid (IDA) . We then introduced such thermoresponsive polymers on cleaned glass coverslips and investigated thermoresponsive lysozyme adsorption. Lysozyme contains only one histigine (His) residue in its amino acid sequences. Thus, IDA-conjugated with nickel ions interacts with His residues through cordinated bonds. Such bonds are easily broken by reducing temperature because hydrated polymer chains. In fact, Lysozyme adsorbed on hydrophobized PIPAAm surfaces with IDA-nickel ion conjugate at 〓℃, while majority of adsorbed proteins spontaneously desorbed from the PIPAAm surfaces without using chelating agents or imidazole adition. This results is promissing for preparation of such surfaces which led us to prepare PIPAAm surfaces with nitrilotriacetic acid (NTA) residues. NTA is currently used in purification of gene products (i.e., proteins) with His-tag. However, such systems still needs chelating agents or imidazole for eluting agent of tagged proteins. Then, we introduced NTA groups through aminolysis of tert-butyl side chains of PIPAAm derivatives on glass surfaces. These surfaces also showed temperature responsive lysozyme adsorption/desorption phenomena in aqueous milieu. In conclusion, we can modulate His-tag protein adsorption/desorption behavior only through temperature modulation. We still needs to improve such systems for application in His tag protein separation, we obtained promissing results in this project. We will continue this work..