Expression and purification of homogenous proteins in Saccharomyces cerevisiae based on ubiquitin-FLAG fusion

Protein Expression and Purification
Adelheid EinhauerAlois Jungbauer

Abstract

The construction of an expression vector for increased expression of cytoplasmic proteins in Saccharomyces cerevisiae is described. To enhance the yield of expressed proteins, fusion of ubiquitin to an octapeptide (a FLAG tag) upstream of the respective model genes was applied. During protein maturation ubiquitin is efficiently removed by yeast autologous hydrolases, generating the FLAG octapeptide at the N-terminus. Fusion proteins were recognized by the specific monoclonal antibody M1 directed against the FLAG tag. The FLAG-tagged proteins were purified to homogeneity by immunoaffinity chromatography using an anti-FLAG M1 agarose. Different model proteins, green fluorescent protein, green fluorescent protein-human lysozyme, green fluorescent protein elongation-initiahon factor 5a, green fluorescent protein-rapamycin-selective 25-kDa immunophilin, and green fluorescent protein-heat shock protein 90 beta have been selected to demonstrate the efficiency of the new vector construct.

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Citations

Dec 3, 2015·Archives of Biochemistry and Biophysics·Ratnika SethiIpsita Roy
Mar 23, 2011·Protein Expression and Purification·Sung-Gun KimJin-Ho Seo
Aug 28, 2015·Molecular Therapy : the Journal of the American Society of Gene Therapy·Rajeev K ChaudharyIpsita Roy
Oct 16, 2007·Analytical Biochemistry·Verena BeckAlois Jungbauer
Jul 11, 2006·Current Opinion in Biotechnology·Liliana FalzonMasayori Inouye
Jul 28, 2005·Journal of Immunological Methods·Shelly Shaki-LoewensteinItai Benhar
Aug 8, 2006·Biotechnology Journal·Alois Jungbauer
Jun 12, 2013·The Biochemical Journal·Kuljit SinghIpsita Roy
Aug 28, 2014·The International Journal of Biochemistry & Cell Biology·Vamshidhar R GadeIpsita Roy
Feb 27, 2014·Biotechnology & Genetic Engineering Reviews·Maria Laura Belladonna, Ursula Grohmann
Jul 19, 2002·Yeast
Jan 7, 2015·Journal of the American Society for Mass Spectrometry·Yelena YefremovaMichael O Glocker
Jun 9, 2020·Biochemical and Biophysical Research Communications·Jung-Ho ShinTobias Dörr
Dec 8, 2021·European Journal of Histochemistry : EJH·Jianyu ZouMinghui Tan

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