Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin

Protein Expression and Purification
Chenyun GuoJingming Yuan

Abstract

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.

References

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Citations

Nov 2, 2007·Applied Microbiology and Biotechnology·Zhonglin ZhaoMin Lin
Jan 27, 2011·Biotechnology Letters·Yifeng Li
May 24, 2006·Bioscience, Biotechnology, and Biochemistry·Zhuanhua WangDan Norback
Oct 10, 2014·Applied Microbiology and Biotechnology·Changhua ShiDavid W Wood
Nov 11, 2014·ACS Chemical Biology·Carrie J MarshallEric V Shusta
Jan 29, 2009·Angewandte Chemie·Christian PiontekCarlo Unverzagt
Jan 12, 2005·Protein Expression and Purification·Carla MorassuttiStefano Marchetti
Sep 17, 2016·Biochemical and Biophysical Research Communications·Tianqing HanYongbo Song
Sep 6, 2006·Acta Biochimica Et Biophysica Sinica·Meng-Jun LuoFang Chen
Feb 1, 2007·The Journal of Biological Chemistry·Hisashi TakahashiMasahiro Iwakura
Jan 16, 2020·International Journal of Molecular Sciences·Angel De La Cruz Pech-CanulAntonio García-Triana
May 13, 2021·Organic & Biomolecular Chemistry·Heather C HayesYu-Hsuan Tsai
Oct 18, 2011·Bioconjugate Chemistry·Kosuke MinamihataNoriho Kamiya
Jul 24, 2012·Bioconjugate Chemistry·Kosuke MinamihataNoriho Kamiya
Aug 9, 2013·Bioconjugate Chemistry·Carrie J MarshallEric V Shusta

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