Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3298
標題: Hydroxyapatite-based immobilized metal affinity adsorbents for protein refolding
以氫氧基磷灰石固定化金屬親和吸附材進行蛋白質復性之研究
作者: 林靜宜
Lin, Ching-Yi
關鍵字: Hydroxyapatite
氫氧基磷灰石
Refolding
Inclusion bodies
GlcNAc 2-epimerase
Immobilized metal ion affinity chromatography
復性
包涵體
差向異構酶
固定化金屬親和層析法
出版社: 化學工程學系
摘要: We overexpressed recombinant GlcNAc 2-epimerase in E. coli as an His-tag recombinant protein. However the expression of this protein in E. coli resulted in the formation of an inactive inclusion bodies(IBs)that intracellularly accumulated. In this paper, we propose a method to recover native and active GlcNAc 2-epimerase from highly insoluble inclusion bodies. This method employing the immobilization of target protein on a hydroxyapatite-Fe3+ surface has been propose to circumvent aggregation of unfolded proteins or folding intermediates because proteins attached to an insoluble carrier may avoid intermolecular aggregation. Under optimal adsorbrd and desorbed conditions, the capacity of hydroxyapatite-based immobilized metal affinity absorbents were 1.11 mg/ g HAP-Fe3+ for total proteins, and the recovery yield was 82%. For on-column refolding, we added 0.5 M L-Arginine and 0.05%PEG to facilitate refolding. A 1.42 ~ 1.59 -fold increased in the total soluble proteins recovery was obtained. In this process, refolding buffer in the presence of 0.5 M L-Arginine was resulting in 2.3 ~ 3.1-fold total activity and 2.1 ~ 2.3-fold specific activity with non additive.
本研究是利用大腸桿菌大量生產基因重組差向異構酶,由於其會在胞內形成大量之包涵體,所以為使包涵體回復成具有天然活性的蛋白質,且避免一般復性程序所導致蛋白質集結的現象,故發展以氫氧基磷灰石固定化金屬親和吸附材進行最適差向異構酶包涵體之復性程序。 以批次實驗探討最適化之吸脫附條件,結果顯示最適化之吸脫附條件下,總蛋白質吸附量為 1.11 mg/g HAP,總蛋白質脫附率可達82%。 再以上述之最適化條件應用於氫氧基磷灰石固定化金屬親和層析管柱進行連續式復性研究,結果顯示於濃度梯度50 ml時,發現停留時間愈長,皆會降低可溶總蛋白質的回收率、總蛋白質活性和比活性,並發現此時所脫附之復性後蛋白質有一部分形成可溶之寡聚體(oligomer)和大多數結構不甚正確之差向異構酶。 於復性程序中,分別添加0.5 M L-Arginine和0.05%PEG,發現添加劑皆有助於增加可溶總蛋白質回收率,皆可增加約1.42倍 ~ 1.59倍之可溶總蛋白質回收率。另外,無論添加L-Arginine的時間皆可有效幫助差向異構酶之復性,且能提升可溶蛋白質之總活性和比活性,與未添加Arginine相比較,分別可得可溶蛋白質之總活性為未添加之2.3 ~ 3.1倍,和比活性為未添加之2.1 ~ 2.3倍。
URI: http://hdl.handle.net/11455/3298
Appears in Collections:化學工程學系所

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