在藥物代謝動力學上，在體內代謝半衰期較短的蛋白藥物可以藉由生物技術融合蛋白的方式與血漿中的蛋白進行結合，以增加代謝的半衰期。人類血清白蛋白 (HSA) 是一個本身就存在於人體中很好的載體，其在血液中的半衰期高達19天，先前的研究也顯示HSA相較於其他血漿中蛋白具有其特別的優勢是一個很好的藥物載體，可以改善小分子蛋白藥物的半衰期。馬來蝮蛇去整合蛋白（Rhodostomin；簡稱Rho）是一個由68個胺基酸所組成的去整合蛋白，並含有六對雙硫鍵。從先前的研究中發現Rho的突變株ARLDDL是特別針對整合蛋白αVβ3具有專一性抑制能力，其IC50值為51.9 nM；然而先前實驗室也成功地將ARLDDL之N端融合了HSA，並且利用P. pastoris表現系統表現，也發現此融合蛋白是針對整合蛋白αVβ3具有專一性抑制能力，同時半衰期也增加了六倍；然而HSA融合蛋白在室溫下三個月後容易被降解，因此為了增加半衰期以及ARLDDL的穩定性，因此我設計了十個可以結合人類血清白蛋白的結合肽(HSABP)，再以三種不同的橋接區域 (linker spacers) 將之以基因重組的方式融合到ARLDDL上。我成功地將這些融合蛋白以P. pastoris表現系統表現這些蛋白，但是其表現量卻低於1 mg/L，在調整優化過後的蛋白產量增加了5至10倍左右。至今，有八個HSABP-ARLDDL融合蛋白進行大量表現並且純化之。從NMR實驗分析的結果顯示，HSABP1-ARLDDL以及HSABP8-ARLDDL融合蛋白之ARLDDL功能區結構有正常摺疊。由非平衡式小區域膠體管柱層析結果得知HSABP1-ARLDDL與HSA的解離平衡常數KD值為397 μM；更進一步由表面電漿共振 (SPR) 實驗的結果顯示其KD值為563 nM。細胞黏著實驗也說明HSABP1-ARLDDL及HSABP8-ARLDDL融合蛋白對於整合蛋白αVβ3也有很好的抑制能力，IC50 分別為28.33 nM及41.21 nM。由這些結果表示HSABPn-ARLDDL融合重組蛋白不但可以與HSA進行結合，而且還保有其ARLDDL區域的3D結構及抑制整合蛋白αVβ3的功能，且橋接區域的大小似乎不影響融合蛋白的功能。這份研究結果也表示HSA結合肽融合去整合蛋白可以將高潛力小蛋白治療藥物結合到HSA，暗示著可能有延長藥物在體內半衰期的可能性。

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of short-lived protein therapeutics. Human serum albumin (HSA) is an endogenous molecule transporter with a half-life of 19 days, and previous studies have indicated that HSA has unique advantages over other plasma proteins as a drug carrier for improving the half-life of small protein drug. Rhodostomin (Rho) is a disintegrin that contain 68 amino acid including six disulfide bonds. Our study shows that Rho mutant ARLDDL is an integrin αVβ3-specific disintegrin with an IC50 value of 51.9 nM. We have successfully expressed HSA-ARLDDL in P. pastoris and found that it retains its activity with a 6-fold increase in half-life; however, HSA was easily degraded at room temperature after three months. To improve the half-life and increase the stability of ARLDDL, I designed ten albumin-binding peptides (HSABP) and used three different linker spacers to fuse them with ARLDDL. They were successfully expressed in P. pastoris; however, their expression levels were less than 1 mg/L. The yield after optimization of their protein production was about 10-fold increase. To date, eight HSABP-ARLDDL fusion protein variants were produced and purified. NMR analysis showed that HSABP1-ARLDDL and HSABP8-ARLDDL fusion protein variants exhibited the correct fold. Nonequilibrium small-zone gel-filtration analysis showed that HSABP1-ARLDDL can bind to HSA with a KD value of 397 μM. Surface plasmon resonance (SPR) showed that the binding of HSABP1-ARLDDL to HSA was determined to have a KD value of 563 nM. Cell adhesion analysis showed that HSABP1-ARLDDL and HSABP8-ARLDDL inhibited integrin αVβ3 with the IC50 values of 28.33 and 41.21 nM. In conclusion, these results suggest that HSABPn-ARLDDL retains its function and 3D structure and can bind to HSA, and the sizes of linker were not affecting their functions. This study demonstrated that HSA-binding peptide-fused proteins designed by this study are potential long-half-life drug candidates.

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