Expression and purification of human perforin by sf9 insect cells

Basic & Clinical Medicine ›› 2020, Vol. 40 ›› Issue (1): 54-59.

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Expression and purification of human perforin by sf9 insect cells

CHEN Feng-ye, LIU Meng-yu, ZHOU Ya-bo, LIANG Xiao-yu, HUANG Bo, XIE Jing^*

National Key Laboratory of Medical Molecular Biology, Department of Immolunology, Institute of Basic Medical Sciences CAMS & School of Basic Medicine PUMC, Beijing 100005, China

Received:2019-10-12 Revised:2019-11-22 Online:2020-01-05 Published:2019-12-27
Contact: ^*xiejing@ibms.pumc.edu.cn

Abstract

Abstract: Objective To optimize the purification method of human perforin. Methods Perforin with His tag was expressed by sf9 insect cells, and then protein affinity chromatography was performed by Ni column device. The physicochemical property and biological activity of the purified protein was analyzed by coomassie brilliant blue staining, silver staining and flow cytometry. Results This method was more convenient and economical than traditional protein purification methods like ion exchange chromatography, molecular sieve and hydrophobic interaction chromatography, which providing new ideas for further research of perforin and purification of other protein. Conclusions Perforin can be expressed by sf9 insect cells and is purified by a simple method with biological activity.

Key words: protein purification, perforin, protein activity, pore-formation

CLC Number:

CHEN Feng-ye, LIU Meng-yu, ZHOU Ya-bo, LIANG Xiao-yu, HUANG Bo, XIE Jing. Expression and purification of human perforin by sf9 insect cells[J]. Basic & Clinical Medicine, 2020, 40(1): 54-59.

References

[1]Lopez JA, Susanto O, Jenkins MR, et al. Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack [J]. Blood, 2013, 121: 2659-2668.
[2]Lowin B, Hahne M, Mattmann C, et al. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas Lytic pathways[J].Nature, 1994, 370:650-652.
[3]Smyth MJ, Kelly JM, Sutton VR, et al. Unlocking the secrets of cytotoxic granule proteins[J].J Leukoc Biol, 2001, 70:18-29.
[4]Russell JH, Ley TJ.Lymphocyte-Mediated Cytotoxicity[J].Annu Rev Immunol,2002, 20:323-370.
[5]Thiery J, Keefe D, Boulant S, et al. Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells[J]. Nat Immunol, 2011, 12:770-777.
[6]Green DR, Llambi F.Cell Death Signaling[J].Cold Spring Harb Perspect Biol, 2015, 7:a006080.doi: 10.1101/cshperspect.a006080.
[7]Chen Ling, Guan-Qing Qiao, Bo-Wen Shuai, et al. Engineering NADH/NAD⁺ ratio in Halomonas bluephagenesis for enhanced production of polyhydroxyalkanoates (PHA)[J]. Metab Eng, 2018, 49, 27-286.
[8]Naneh O, Zavec AB, Pahovnik et al. An optimized protocol for expression and purification of murine perforin in insect cells[J]. J Immunol Methods, 2015, 426:19-28.
[9]Yarchoan M, Johnson BA, Lutz ER, et al. Targeting neoantigens to augment antitumour immolunity [J]. Nat Rev Cancer, 2017, 17: 209-222.
[10]Trapani JA, Smyth MJ. Functional significance of the perforin/granzyme cell death pathway [J]. Nat Rev Immunol, 2002, 2: 735-747.
[11]Froelich CJ, Turbov J, Hanna W. Human Perforin: Rapid Enrichment by Immolobilized Metal Affinity Chromatography (IMAC) for Whole Cell Cytotoxicity Assays[J].Biochem Biophys Res Commun, 1996, 229:44-49.
[12]Thiery J, Walch M, Jensen DK, et al. Isolation of cytotoxic T cell and NK granules and purification of their effector proteins[J]. Curr Protoc Cell Biol, 2010, 47:Unit3.37.doi: 10.1002/0471143030.cb0337s47.

Expression and purification of human perforin by sf9 insect cells

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