The solubility and stability of recombinant proteins are increased by their fusion to NusA

Biochemical and Biophysical Research Communications
Valeria De MarcoArio de Marco

Abstract

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.

References

Apr 1, 1991·Protein Expression and Purification·C F FordC E Glatz
May 1, 1994·Trends in Biotechnology·P A NygrenM Uhlén
Aug 19, 1999·Protein Science : a Publication of the Protein Society·R B Kapust, D S Waugh
Oct 3, 1999·Biotechnology and Bioengineering·G D DavisR G Harrison
Jul 14, 2001·Molecular Biology of the Cell·N TolonenJ Krijnse Locker
Sep 26, 2001·Protein Expression and Purification·Y NominéG Travé
Jul 2, 2003·EMBO Reports·Valeria De MarcoAndreas Hoenger

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Citations

Jun 10, 2011·Applied Microbiology and Biotechnology·Guoqiu WuHanmei Xu
May 1, 2010·Biotechnology Letters·Pieranna ChiarellaArio de Marco
Dec 24, 2008·Molecular Biotechnology·Xiaoping WuXiaokun Li
Jun 18, 2005·Biochemical and Biophysical Research Communications·Pierre DouetteFrancis E Sluse
Feb 12, 2010·Nucleic Acids Research·Nithya BabuRajendranPrasanna R Kolatkar
Nov 20, 2009·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Nithya BaburajendranPrasanna R Kolatkar
Aug 3, 2011·Microbial Cell Factories·Esther VazquezAntonio Villaverde
Dec 3, 2008·Microbial Cell Factories·Spela PeternelRadovan Komel
Feb 2, 2013·PloS One·Henrik NauschHeike Mikschofsky
Jun 16, 2011·International Journal of Molecular Sciences·Krishna M P DasSriram Padmanabhan
Apr 1, 2014·Protein Expression and Purification·Karolina PeciakEmmanuelle Laurine
Nov 17, 2012·Journal of Microbiological Methods·Gro Anita StamsåsDaniel Straume
May 12, 2010·Protein Expression and Purification·Zhongyuan WangXuexun Fang
Mar 17, 2010·Biochemical and Biophysical Research Communications·Marta PasekPradman K Qasba
Sep 11, 2008·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Paula C PohlAoi Masuda
Feb 1, 2008·Journal of Molecular Biology·Anna GribunWalid A Houry
Jan 8, 2008·Journal of Molecular Biology·Ralf JauchPrasanna R Kolatkar
Jan 14, 2010·The FEBS Journal·Anissa BoumlicGeorges Orfanoudakis
Sep 10, 2005·Protein Expression and Purification·Michelle K M ChowStephen P Bottomley
Jun 1, 2005·Trends in Biotechnology·David S Waugh
Aug 9, 2005·Protein Expression and Purification·Tauseef R ButtMichael R Mattern
Jul 16, 2015·Preparative Biochemistry & Biotechnology·Jun ZhangZandong Li

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