研究能自發斷裂之胜？並應用於提高蛋白質純化之效能

Abstract

蛋白質純化一般涉及多個與管柱層析有關的操作，例如離子交換、疏水性交互作用和凝 膠過濾等。純化蛋白質的最佳方式是利用與具有特定官能基之凝膠進行親和性結合來達 到分離及純化的目的，這類具有特定官能基的凝膠管柱層析法包括，例如，鎳管柱層析、 榖胱氨肽改質之管柱層析、麥芽糖改質之凝膠管柱層析或其他等等。但是，目前盛行的 這類蛋白質管柱層析法尚有許多缺陷有待克服，例如，通常所獲得的是一種融合蛋白， 因此後續會需要用到蛋白酶來去除不要的融合部位，或是需要用到可進行親和性結合但 價格高昂的凝膠。有鑑於此，相關領域亟需提出一種可用來產生純化蛋白質且不需用到 蛋白酶處理之改良的方法和/或系統，此種改良的方法和/或系統本身易於使用、具經濟 效益且可在不犧牲標的蛋白本身功能的情況下，用來大量生產出純化的標的蛋白質。 本實驗室最近建構一含有幾丁質結合蛋白質(CBP)和(EAAAK)5 胜肽連接子的表現系 統，並意外發現經幾丁質管柱純化所得之融合蛋白上，其(EAAAK)5 胜肽連接子有自發 斷裂的特性。此系統可利用廉價幾丁質管柱且不需使用蛋白酶即可得重組之目標蛋白， 極適合發展量產高階蛋白之應用。然而此系統仍存在許多學理與技術應用待進一步研究: 1. 如何加強並操控胜肽連接子自我斷裂之速度 2. 如何最適化CBP 與幾丁質於不同pH 下之結合力 3. 如何克服產生涵胞體(inclusion body)的問題 4. 驗證CBP/auto-cleavage-linker 載體於其他表現系統，如Pichia pastoris 表現系統之可 行性。 為研究上述問題，我們將針對一系列可形成α-helix 結構之重複性五胜肽，如(EAAAK)5, (EAAAR)5, (EGGGK)5, 和 (EGGGR)5 等進行胜肽和蛋白質層級之自發斷裂研究。將應 用基因改質技術局部調整CBP 與幾丁質結合區的結構，並研究其突變後之結合力。為 克服重組蛋白之溶解度(或形成涵胞體)之問題，我們將於新建之載體上引入大腸桿菌一 periplasmic 蛋白osmY，osmY 最近被發現可有效將與其融合之蛋白攜出大腸桿菌外。利 用此載體可望將許多溶解度不佳之人體蛋白質直接表現於胞外，並結合幾丁質管柱和胜 肽連接子自發斷裂的特性純化此等蛋白質。本計畫將先鎖定人體金屬蛋白酵素13 和15 (matrix metalloproteinase 13 和15)之表現，其他如轉醣酵素亦可嚐試。成功大量表現此 等酵素將對許多疾病之研究有重大幫助。此外，我們亦將應用這類型表現載體於它種表 現系統中，如Pichia pastoris。 本計畫預計執行三年，年度工作大項如: 第一和第二年 1. 研究(EAAAK)5、(EAAAR)5、(EGGGK)5和(EGGGR)5胜肽之自發斷裂化學反應機構 2. 建構含有(EAAAK)5、(EAAAR)5、(EGGGK)5和(EGGGR)5胜肽連接子與CBP之各式表 現載體 3. 研究含各種胜肽連接子之融合蛋白質的自發斷裂性質和斷裂速度之操控 4. 以蛋白質基因工程改進CBP 對幾丁質之親和力 5. 應用和評估此類新穎表現載體於大量蛋白質純化之可行性 第二和第三年 The second year – the third year 1. osmY之基因選植 2. 重建含osmY、CBP、自發斷裂胜肽連接子之表現載體 (命名為pOsCL載體) 3. 應用新式表現載體製備和純化MMP13 和 MMP15，並研究攜出蛋白之自發斷裂性質 4. MMP13 和 MMP15 之活性分析 5. 含有自發斷裂胜肽連接子之新式表現載體於 Pichia pastoris 表現系統的功能

Protein purification generally requires many steps of column chromatography that typically involve ion-exchange, hydrophobic-interaction and gel-filtration separations. More sophisticated purification of protein might be achieved through an application of affinity binding on a functionalized gel such as a nickel column, glutathione-modified column, maltose-modified gel column or others. Of several drawbacks existing in these methods, fusion proteins are commonly obtained, protease-digestion might be necessary to remove the fusion moiety; a costly gel is employed for affinity binding, etc. Very recently, we constructed an expression vector, derived from pREST, that composed the gene of the chitin-binding protein (CBP) and the nucleotide sequence of the (EAAAK)5 peptide linker following restriction sites for target gene insertion. Fusion proteins can be expressed with E. coli and purified with a chitin column. Interestingly, the (EAAAK)5 linker is shown to possess a pH-dependent auto-cleavage feature. In the range pH 6-7, the target protein becomes automatically released from the fusion protein without proteolytic treatment. The system is potential useful for protein purification in large–scale if the following issues can be solved: (1) the rate of auto-cleavage of the linker needs to be enhanced and controlled; (2) the binding affinity of CBP towards chitin needs to be optimized within pH 6-7; (3) the situation of recombinant protein forming inclusion body in E. coli needs to be improved; (4) different expression system and host need to be evaluated. To study the above subjects, we will investigate the kinetics and reaction mechanism of auto-cleavage using a series of repeated helix-forming peptide such as (EAAAK)5, (EAAAR)5, (EGGGK)5, (EGGGR)5 at peptide level and also at protein level. The chitin-binding region of CBP will be engineered to study the binding affinity at various pH conditions. In order to possibly overcome the problem of inclusion body, we will introduce the OsmY (a perilasmic protein of E. coli) as the carrier for those proteins, such as human matrix metalloprotease 13 and 15, which have been failed to be produced as soluble form in E. coli. Other expression system, such as Pichia pastris will be tested with the newly developed CBP system. The listed subjects will be performed in the following 3 years: The first year – the second year: 1. Study the reaction mechanism of auto-cleavage of (EAAAK)5, (EAAAR)5, (EGGGK)5, and (EGGGR)5 peptide. 2. Construction of expression vectors with (EAAAK)5, (EAAAR)5, (EGGGK)5, and (EGGGR)5 linker 3. Study the auto-cleavage property of the above repeated peptide in the fusion protein 4. Genetic engineering of the chitin-binding domain in CBP and Study the chitin-binding property of CBP mutants 5. Evaluation the newly designed vector in large scale protein purification The second year – the third year: 1. Cloning and expression of osmY 2. Construction of the expression vector containing osmY, CBP and the auto-cleavage linker (the vector is designated pOsCL) 3. Construction of MMP13 and MMP15 in pOsCL 4. Expression and purification of MMP13 and MMP15 5. Study the properties of protein secretion and auto-cleavage feature of various linkers 6. Construct and evaluate the newly designed vectors in Pichia pastoris expression system 7. Study the auto-cleavage feature of the recombinant protein from Pichia pastoris expression system