整合蛋白是一群位於細胞表現的異質雙體，調整許多細胞步驟，如生長、死亡、黏著、移動。馬來亞腹蛇蛋白為一種有淺力的血小板凝集抑制劑，且屬於去整合蛋白家族一元。馬來亞腹蛇蛋白由68個胺基酸，其序列上帶有48PRGDM53P及6對雙硫所建構而成。我們實驗室先前由就已經成功利用嗜甲醇酵母菌表現馬來亞腹蛇蛋白，且利用它設計出對整合蛋白αvβ3專一性的藥物。本篇研究內容，我主要是做最佳化嗜甲醇酵母菌發酵槽條件，增加嗜甲醇酵母菌表現馬來亞腹蛇突變蛋白質HAS-ARLDDL之產量。比較搖瓶及發酵槽生產馬來亞腹蛇突變蛋白上清液中總蛋白含量，可以得知利用搖瓶產量約為 100 mg/L，而藉由最佳化pH值、溫度、培養基成分、和發酵槽中氧氣供給程度，能使發酵槽上清液總蛋白產量達到 900 mg/L，然而分別純化後產量分別為 30 mg/L 及 170 mg/L。檢視搖瓶及發酵槽兩者純化過程中的回收率分別為30%及17%，似乎是由於發酵槽有偏高的錯誤摺疊蛋白從發酵過程中產生出來。在先前研究我們發現馬來亞腹蛇突變蛋白其連結區域帶為39KKKRTICR47I序列會影響其結合能力及選擇在整合蛋白α5β1, αvβ3, and αIIbβ3，本篇研究中，我已經成功表現三株在連結區域為39KKKRT並含R46A、I47R、R46A/I47R之馬來亞腹蛇突變蛋白及一株在連結區域為39SRAGK並含R46A之馬來亞腹蛇突變蛋白，藉由分析血小板凝集及細胞黏著實驗，結果顯示在連結區域為39SRAGK並含R46A之馬來亞腹蛇突變蛋白會對血小板凝集及整合蛋白αIIbβ3、αvβ3、及 α5β1的活性，分別為 5.4、7.6、2.2、及5.6倍下降。此外連結區域序列為39KKKRT並含R46A之馬來亞腹蛇突變蛋白也會對血小板凝集及整合蛋白αIIbβ3、αvβ3、及 α5β1的活性，分別為 4.1, 24.8, 1.8,及1.8倍下降。而含序列39KKKRT並含 I47R之馬來亞腹蛇突變蛋白能增加對整合蛋白αvβ3、及 α5β1的活性，分別為3、7倍上升。有趣的是，我們發現突變Rho蛋白含39KKKRTIC46AR-ARGDN53P-67Y68H序列對抑制整合蛋白αIIbβ3、αvβ3、及 α5β1之IC50值分別為956.0 nM、5.7 nM、6.6 nM。這項發現可以去除Rho蛋白對整合蛋白αIIbβ3之抑制能力。總體而言，這些突變Rho蛋白之功能研究結果能夠提供有用資訊設計對整合蛋白專一的去整合蛋白，而最佳化表現HAS-ARLDDL蛋白之嗜甲醇酵母菌發酵槽條件，能夠提供一些基本資訊用來表現對整合蛋白專一的去整合蛋白

Integrins are a family of heterodimeric receptors and modulate many cellular processes, including growth, death, adhesion, and migration. Rhodostomin (Rho) is a potent platelet aggregation inhibitor and belongs to the family of disintegrins. Rho consists of 68 amino acids with a 48PRGDM53P motif and six disulfide bonds. In previous study we expressed Rho in Pichia pastrois (P. pastoris) and used it as the scaffold to design integrin αvβ3-specific drugs. In this study I optimized the fermentation culture condition to increase the protein production of Rho mutants expressed in P. pastoris. The protein production of Rho mutants using shake-flask culture is ~100 mg/L. I increased the production of a Rho mutant, HSA-ARLDDL, up to 9-fold with a yield of ~900 mg/L by optimizing pH, temperature, medium composition, and oxygen level of the fermentation process. The final yields of proteins produced from shake-flask and fermentation cultures after purification were 30 mg/L and 170 mg/L, respectively. The recovery of proteins produced from shake-flask and fermentation cultures after purification were 30% and 17%, respectively. It is likely due to higher mis-folding proteins produced from the fermentation process. In our previous study we found that Rho mutants with a 39KKKRTICR47I linker sequence affected their binding affinity and selectivity to integrins α5β1, αvβ3, and αIIbβ3. Therefore, I have successfully expressed three Rho mutants with the 39KKKRT linker sequence (R46A, I47R, and R46A/I47R mutants) and one mutant with the 39SRAGK linker sequence (R46A mutant). The analysis of platelet aggregation and cell adhesion assays showed that the R46A mutant with the 39SRAGK linker sequence exhibited 5.4-, 7.6-, 2.2-, and 5.6-fold decreases in their inhibitory activity to platelet aggregation and integrins αIIbβ3,αvβ3, and α5β1, respectively. In addition, the R46A mutant with the 39KKKRT linker sequence exhibited 4.1-, 24.8-, 1.8-, and 1.8-fold decreases in their inhibitory activity to platelet aggregation and integrins αIIbβ3, αvβ3, and α5β1. In contrast, the I47R mutant with the 39KKKRT linker sequence exhibited 3- and 7-fold increases in their inhibitory activity to integrins αvβ3 and α5β1, respectively. Interestingly, we found that Rho mutant containing the 39KKKRTICARARGDN53P sequence inhibited integrins αIIbβ3, αvβ3, and α5β1 with the IC50 values of 956.0, 5.7, and 6.6 nM, respectively. The finding indicated that this mutant can abolish its activity to integrin αIIbβ3. The mutational study on Rho will provide new insight into design potent integrin-specific disintegrins, and the optimization of fermentation condition of HSA-ARLDDL will serve the basis to produce integrin-specific disintegrins expressed in P. pastoris.

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