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| Development of A Bifunctional Tag Used for Affinity Purification and Transmembrane Transport of Drug Proteins |
GUO Li-cheng1,CAO Xue-wei1,FU Long-yun2,WANG Fu-jun2,3,ZHAO Jian1,**( ) |
1 State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China
2 Zhejiang FONOW Medicine Co. Ltd,Dongyang 322100,China
3 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China |
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Abstract 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.
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Received: 13 January 2020
Published: 23 June 2020
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Corresponding Authors:
Jian ZHAO
E-mail: zhaojian@ecust.edu.cn
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