本論文主要針對先前以壓電生物感測晶片與微陣列細胞生物晶片，對鞭毛隨機胜肽組合庫篩選出對streptavidin (SAv)具有較強親和性之四段不同胜肽鏈作進一步探討。首先將此四段胜肽分別以基因重組技術接合於N-carbamoyl-D-amino acid amidohydrolase（DCase）酵素之C端，以大腸桿菌BL21(DE3)成功表現出DCase-aJ21、DCase-bJ13、DCase-hM8、DCase-strepII等融合蛋白，並進行活性分析，而後再分別以SAv凝膠與修飾有SAv之壓電晶片注流式系統對重組蛋白進行親和性分析。由活性分析之結果可得知DCase-hM8的活性不受融合標籤之影響，其比活性甚至高於原生種，約為4.3 U/mg。爾後於SAv凝膠分析中，以蛋白質電泳以及濃度分析確認此些酵素確實與SAv具有親和性；其中與SAv凝膠親和性最強之融合酵素為DCase- bJ13，其Kd值約為4.9 μM；而其與SAv壓電晶片之Kd值則低達1.07 μM。最後綜觀兩分析系統之結果，可確知DCase-bJ13與SAv之親和性最高，因此bJ13胜肽最適合當作SAv親和性標籤。

This study continues the previous researches where biopanning, microarrays, and piezoelectric (PZ) biochips were sequentially used to screen four streptavidin (SAv) affinity peptides from an E.coli flagellum-displayed random peptide library. Those four peptides were individually fused to the C terminus of N-carbamoyl-D-amino acid amidohydrolase (DCase) and expressed in E. coli BL21 (DE3), yielding four fusion proteins, DCase-aJ21, DCase-bJ13, DCase-hM8 and DCase-strepII, respectively. The proteins were subsequently each assayed for their enzymatic activities and SAv affinities using SAv gels and flow-injection SAv-PZ biochips. The DCase-hM8 enzyme showed a specific activity of 4.3 U/mg, which is the most active one among all DCase and DCase fusion proteins. The SDS-PAGE and protein concentration assays conformed the SAv-binding affinities of all fusion proteins, with DCase-bJ13 possessing the highest affinity with dissociation constants (Kd) of 4.91 μM when analyzed using SAv gels and of 1.07 μM when analyzed using SAv-PZ biochips. It is concluded that DCase-bJ13 has the best affinity with SAv, and therefore is the most promising peptide as a SAv affinity tag.

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