一种用于药物蛋白亲和纯化和跨膜转运的双功能标签的开发

目的:开发一种既能用于亲和纯化目标蛋白,又可介导不能自主进入细胞的药物蛋白跨膜转运到细胞内发挥活性的双功能标签。方法:从已有文献资料中挑选四种富含碱性氨基酸的钙调蛋白结合肽(calmodulin binding peptide,CBP),将其与绿色荧光蛋白(EGFP)融合表达,然后采用与钙调蛋白(calmodulin,CaM)亲和结合过程来筛选与CaM具有最高亲和力的CBP;随后采用荧光显微镜检测、激光共聚焦显微镜检测以及流式细胞术等技术来分析测定和比较候选CBP序列将EGFP重组蛋白自主转运进入细胞的能力。最后将筛选到的新型CBP双功能标签与凋亡蛋白融合表达,考察其与CaM亲和结合后纯化重组凋亡蛋白的能力,以MTT法分析此重组蛋白进入肿瘤细胞抑制生长的能力。结果:通过CaM-CBP亲和层析筛选出与CaM具高有亲和力的三种CBP序列;从重组蛋白胞内荧光检测结果得知,带有野生型骨骼肌肌球蛋白轻链激酶CBP序列(MLCK)的重组EGFP蛋白具有最佳跨膜转运效率,且显著高于来源于艾滋病毒的经典穿膜肽TAT的穿膜效率。以此MLCK新型双功能标签成功地通过CaM-CBP亲和结合纯化得到重组凋亡蛋白,并可将重组凋亡蛋白转运进入细胞内发挥抗肿瘤作用。重组凋亡蛋白对MGC-803、H460、HeLa三种肿瘤细胞生长的24h半抑制浓度(IC50)分别为:1. 18μmol/L、1. 23μmol/L、1. 23μmol/L。结论:筛选得到一种新型双功能标签MLCK,其可通过与CaM高亲和作用进行亲和纯化;同时标签本身还具有和典型穿膜肽一样的高效跨膜转运功能,可将药物蛋白自主转运进入细胞,发挥药物的生物活性。因此,新型双功能标签既可用于药物蛋白的亲和纯化,又兼具体内跨膜运输作用,可广泛用于各种新型药物的开发。

Development of A Bifunctional Tag Used for Affinity Purification and Transmembrane Transport of Drug Proteins

Objective: To develop a bifunctional tag which can be used for affinity purification of target proteins, and can carry some drug proteins that cannot enter the cell autonomously across cell membrane to exert activity.Methods: Four types of calmodulin binding peptide (CBP) rich in basic amino acids are selected from the literature as candidate tags for fusion expression with green fluorescent protein (EGFP). The CBP with the highest affinity to CaM is screened by CaM affinity binding process. Subsequently, the capacity of the candidate CBP sequences to autonomously transport EGFP recombinant proteins into cell is analyzed and determined by fluorescence microscopy, laser confocal microscopy and flow cytometry. Finally, the novel CBP bifunctional tag is screened then fused and expressed with Apoptin, and its ability to purify recombinant Apoptin is examined after binding with CaM. The ability of this recombinant protein to enter tumor inhibiting cell growth is analyzed by MTT method.Results: Three novel CBP sequences with high affinity to CaM are screened out by CaM-CBP affinity chromatography. The intracellular fluorescence detection results of the recombinant protein showed that the CBP sequences derived from wild-type skeletal muscle myosin light chain kinase (named MLCK) has the best transmembrane efficiency to carry EGFP into cells, and its transportation capacity is much higher than the classic cell-penetrating peptides TAT derived from HIV. The recombinant Apoptin is purified by CaM-CBP affinity chromatography with the affinity tag MLCK and can be transported into cells to exert an anti-tumor activity. Furthermore,MTT assay results show that the 24-hour 50% inhibitory concentrations (IC₅₀) of the recombinant Apoptin on three tumor cells MGC-803, H460, and HeLa are 1.18μmol/L, 1.23μmol/L, and 1.23μmol/L, respectively, which shows that the Apoptin retains partial anti-tumor activity.Conclusion: A novel calmodulin-binding peptide, MLCK, is screened out which can be used in affinity purification due to its high affinity with CaM. Simultaneously, it has high-efficiency transmembrane function which is similar to typical cell-penetrating peptides, and can carry drug proteins into cells autonomously to exert the biological activity of drugs. Therefore, the novel bifunctional tag can be used for both affinity purification of drug proteins and cell membrane transportation, thus it can be widely used in the development of various new drugs.