Preparation of high affinity molecularly imprintedpolymers selective for denatured creatine kinase andtheir application to the extraction of native anddenatured proteins

• Main Authors: Yi-chen Chen, 陳怡臻
• Other Authors: Chung-yih Wang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/76482411162610156241

碩士 === 大同大學 === 生物工程學系(所) === 97 === The inter-relationship of protein structure and function is of interest to many researchers; however, formal academic study of denatured proteins, arising e.g. from genetic mutations, has until now been limited. Of critical significance are the degenerative processes that lead to the conversion of the α-helix to the β-sheet, and the generation of amyloid plaques in for example: Alzheimer’s disease, Parkinson’s disease and degenerative conditions associated with prions. Here a simple synthetic approach based on micro- contact imprinting has been used to form imprints of the secondary structure of creatine kinase (CK), denatured by treatment with SDS. The imprinted materials, formed with MMA and PEG400DMA, in a volume ratio of 5:95, have been demonstrated to be able to separate denatured CK from its native from. Using NaOH/trypsin, a template extraction-efficiency, i.e. denatured CK removal from the film, of 90% was obtained. Subsequent re-binding of the template, i.e. denatured CK, was typically 70%, (template solution 3.5 ×10 -7 mol, MIP surface area 1.69 cm2), with an imprinting efficiency of 9.7. Evaluation of the imprinted polymers in non-competitive re-binding experiments with native and denatured proteins, showed re-binding of 68.3%, 95.3%, 92.3% and 62.4% to the native forms of CK, IgG, HSA and myoglobin respectively; while the respective binding to denatured forms of IgG, HSA and myoglobin was 94.1%, 92.7%, 71.7%. In a competitive binary system, using the denatured forms of: IgG, HSA and myoglobin; the selectivity was 98.7%, 96.8% and 63.8%, respectively. The method used has been shown to be a successful approach for the formation of high-affinity materials able to separate denatured CK from its native form and from other denatured proteins. Such materials may find future applications in practical sensing devices.