開發暫時性蛋白質錨定系統可用於高通量分選具最高蛋白質產量之細胞

Development of a transiently protein-anchored system for high-throughput sorting of the most productive protein-producing cell

江怡宣(I-Shiuan Chiang)

指導教授 ： 鄭添祿

高雄醫學大學/生命科學院/生物醫學暨環境生物學研究所/碩士(2012年)

摘要

利用哺乳動物細胞生產重組蛋白質藥物已是目前的生技醫藥發展的主流；然而，現今仍缺乏一套準確且可高通量篩選具最高蛋白質產能細胞株的方法。為了達成此目的，我們開發一套創新的暫時性膜蛋白錨定技術，搭配流式細胞分選技術，即可高效率地分選出高產能之細胞株。我們利用furin蛋白酶切位片段(RAKR)連接人類抗表皮生長因子抗體(anti-EGFR antibody)與老鼠B7胞外-穿膜蛋白序列 (B7)，藉由調控furin蛋白酶活性以控制anti-EGFR抗體分泌或表現於細胞膜上。將基因轉殖入人類胚胎腎細胞HEK-293T可穩定分泌anti-EGFR抗體；當加入furin 抑制劑(Dec-RVKR-CMK)阻斷furin 蛋白酶活性時，anti-EGFR抗體會暫時地轉化成膜抗體蛋白，伴隨著抗體分泌量下降之情形；反之，當移除furin 抑制劑後，HEK-293T/anti-EGFR細胞可恢復分泌抗體之能力。我們進一步分選出23組HEK-293T/anti-EGFR單株細胞，統計其膜抗體表現量與抗體分泌量之關係，結果顯示兩者具有高度的正相關性(R2=0.91)。更重要的是，此技術亦可應用於分選高度表現溶酶體重組蛋白之細胞株；以Dec-RVKR-CMK 阻斷furin蛋白酶活性，可使溶酶體內部分的穿孔素-顆粒溶解酶(perforin-granzyme)重組蛋白質暫時地表現於細胞膜上；其膜表現量與溶酶體內表現量亦呈現高度正相關性(R2=0.94)。我們相信此創新的暫時性膜蛋白錨定技術能夠快速且高效率地分選出具高產能的細胞株，有效降低重組蛋白藥物的生產成本，提升其臨床醫療價值。

關鍵字

暫時性蛋白質錨定技術 ； 流式細胞分選技術 ； 高產能細胞 ； 重組蛋白藥物

並列摘要

Using mammalian cells to produce recombinant protein drugs has become the global mainstream of current biopharmaceutical markets. However, an accurate and high-throughput method for the effective selection of high-producing cells still awaits development. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of high-producing clones. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (anti-EGFR antibody) and the extracellular-transmembrane domains of the mouse B7-1 antigen. Thus, we can control the anti-EGFR antibody as a secreted antibody or a membrane-anchored protein by modulating the furin protease activity. Transfected HEK-293T cells stably secrete anti-EGFR antibodies. When adding the furin inhibitor (Dec-RVKR-CMK), the anti-EGFR antibody temporarily converted to a membrane-anchored protein, following the descent of secreted antibodies. On the contrary, after removing the furin inhibitor, HEK-293T/anti-EGFR cells restore antibody-secreting ability. We further selected and calculated 23 monoclonal HEK-293T/anti-EGFR cells, demonstrating a high correlation (R2=0.91) between secretion level and surface expression level of anti-EGFR antibodies. Moreover, this method is also suitable for efficient selection of high-producing cells for lysosomal recombinant proteins. Using Dec-RVKR-CMK to block furin activity leads a small amount of the perforin-granzyme recombinant protein temporarily expressing on cell surface, allowing us accurately predicting the relative expression level of lysosomal perforin-granzyme proteins in each clone (R2=0.94). The novel transiently protein-anchored system can easily and efficiently select high-producing cells, reducing the cost for the production of biopharmaceuticals, further improving their medical values.

並列關鍵字

transiently protein-anchored system ； fluorescence activated cell sorting (FACS) ； high-producing cell ； recombinant protein

參考文獻

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