目前使用大腸桿菌系統生產重組蛋白質常遭遇可溶性不足或重組胜肽分子量過小不易回收純化等問題。本研究運用一段可提升目標重組蛋白於大腸桿菌系統可溶性的胜肽標誌及可幫助重組蛋白純化的親和性標誌―Starch binding domain (SBD)提升具功能性重組蛋白質之可溶性與回收率。本研究以具有醣胺聚糖(Glycosaminoglycan、GAG)結合能力的人類嗜酸性白血球陽離子蛋白(Eosinophil cationic protein、ECP)及由ECP序列中核心GAG結合區域衍生之無毒性細胞穿透胜肽(Cell penetrating peptide、CPPecp)為標的，設計、表現與純化重組蛋白質。以往利用細菌表現ECP時會形成不可溶之包涵體(Inclusion body)，故至今尚無有效利用大腸桿菌系統生產可溶性ECP的案例。本研究首先以胜肽標誌修飾ECP胺基端生產重組ECP蛋白，成功表現並純化可溶性重組ECP，初步蛋白質功能性分析顯示此重組ECP仍保有GAG 結合活性。以化學法合成胜肽有序列依賴性，在回收率與純度方面受到限制，本研究第二部分將SBD修飾至CPPecp胺基端並以胜肽標誌連接兩者，成功表現並純化重組CPPecp蛋白，功能性分析結果顯示此重組CPPecp仍具有GAG 結合與細胞穿透能力。因此，本研究之胜肽標誌與SBD有效幫助功能性重組蛋白及重組胜肽在大腸桿菌系統中表現可溶性蛋白質及後續純化分析，未來會測試更多目標蛋白以確認結合胜肽標誌及SBD作為提升重組蛋白可溶性的親和性標誌之應用方法。

Currently insufficient protein solubility and peptide recovery remains a major bottleneck in expression of recombinant proteins and peptides in E. coli system. Our laboratory antecedently identified a solubility enhancing peptide which facilitates expression of soluble recombinant proteins in E. coli, and a starch binding domain (SBD) which has become a well-developed affinity purification tag. In this study, human eosinophil cationic protein (ECP), a glycosaminoglycan (GAG) binding protein which has never been isolated with a soluble form in E. coli system and a 10 amino-acid peptide, non-cytotoxic as well as multifunctional cell-penetrating peptide (CPPecp) holding the core GAG binding region of ECP, were taken as examples to assess the solubility enhancing peptide and SBD in expression and purification of insoluble protein and functional peptide in E. coli system. By fusing an N-terminal solubility enhancing peptide, soluble recombinant ECP could be expressed and purified without time-consuming refolding process from insoluble inclusion bodies. Preliminary functional studies revealed that the N-terminal fusion of recombinant ECP remained its GAG binding activity. For CPPecp, an N-terminal SBD was fused to CPPecp spaced with an internal solubility enhancing peptide as recombinant SBD-CPPecp was successfully expressed and purified with GAG binding and cell-penetrating activities. These results suggested that the solubility enhancing peptide and SBD aid in expression and purification of functional recombinant ECP and CPPecp, which significantly facilitate further application of the solubility enhancing peptide and SBD as effective fusion partners in near future.

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