A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

BMC Biotechnology
Lisa D CabritaStephen P Bottomley

Abstract

In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA) and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease) of the fusion protein to yield native protein. These vectors ar...Continue Reading

References

Oct 25, 1990·Nucleic Acids Research·C Aslanidis, P J de Jong
Nov 15, 2000·Genome Research·J L HartleyM A Brasch
Feb 22, 2002·Acta Crystallographica. Section D, Biological Crystallography·Matthew H BucherDavid S Waugh
Oct 16, 2002·The Journal of Biological Chemistry·Jason PhanDavid S Waugh
Jan 23, 2003·Protein Science : a Publication of the Protein Society·Ailong Ke, Cynthia Wolberger
Jun 26, 2003·Protein Science : a Publication of the Protein Society·Douglas R SmythBostjan Kobe
Dec 31, 2003·Methods : a Companion to Methods in Enzymology·Phillip I BirdStephen P Bottomley
Jul 10, 2004·Structure·Jeffrey A Chao, James R Williamson
Sep 1, 2004·Biochemical and Biophysical Research Communications·Valeria De MarcoArio de Marco
Oct 27, 2004·Journal of Structural and Functional Genomics·Esmeralda A WoestenenkHelena Berglund
Oct 27, 2004·Biotechnology Annual Review·Lisa D Cabrita, Stephen P Bottomley
Mar 23, 2005·Nature Methods·Ashley M BuckleStephen P Bottomley
May 26, 2005·Protein Expression and Purification·F William Studier
Aug 30, 2005·Proteomics·Ulrike KorfAnnemarie Poustka

❮ Previous
Next ❯

Citations

May 25, 2010·Applied Microbiology and Biotechnology·Sun Mee HongTakahiro Kusakabe
Oct 1, 2011·Molecular Biotechnology·Alexander ZawairaDenis Chopera
Feb 1, 2012·Asian Pacific Journal of Tropical Biomedicine·Orawan Khow, Sunutcha Suntrarachun
Oct 25, 2008·Chemical Society Reviews·Min-Hsiung Pan, Chi-Tang Ho
Jun 8, 2007·Microbial Cell Factories·Asgar ErginThomas Adam
Jan 20, 2007·Microbial Cell Factories·Keith Al-HasaniBen Adler
Jul 31, 2013·AMB Express·Felix JakobUlrich Schwaneberg
Jun 16, 2011·International Journal of Molecular Sciences·Krishna M P DasSriram Padmanabhan
Dec 15, 2015·Molecular Biology and Evolution·Brantley D ColemanMikel Garcia-Marcos
Feb 11, 2015·Nature Structural & Molecular Biology·Christopher H S AylettNenad Ban
Mar 24, 2017·Protein Expression and Purification·Jennifer Konczal, Christopher H Gray
Nov 21, 2007·Protein Expression and Purification·Nuoyan ZhengQi Xie
Dec 19, 2006·Journal of Molecular Biology·Glenn A PowersStephen P Bottomley
Feb 24, 2007·Protein Expression and Purification·William T JonesTaha Al Samarrai
Nov 15, 2017·Proceedings of the National Academy of Sciences of the United States of America·Anthony LeymeMikel Garcia-Marcos
Jun 30, 2018·Preparative Biochemistry & Biotechnology·Elaheh KhosrowabadiKhosro Khajeh
Feb 3, 2018·Scientific Reports·Li YangStephen P Bottomley
Oct 2, 2007·Protein Science : a Publication of the Protein Society·Lisa D CabritaStephen P Bottomley
Jul 29, 2009·Biotechnology Progress·Hui-Min WangPo-Huang Liang
Dec 20, 2014·Microbial Cell Factories·Shubhashree MahalikKrishna J Mukherjee
Nov 17, 2010·Applied Microbiology and Biotechnology·Sangkyu ParkKyoungwhan Back
Jul 4, 2019·Molecular Biology of the Cell·Arthur Marivin, Mikel Garcia-Marcos
Apr 18, 2013·Angewandte Chemie·James T PayneJared C Lewis

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
solubility assay
solubility
NMR
circular dichroism
Assay

Software Mentioned

pLIC

Related Concepts

Related Feeds

Bioinformatics in Biomedicine

Bioinformatics in biomedicine incorporates computer science, biology, chemistry, medicine, mathematics and statistics. Discover the latest research on bioinformatics in biomedicine here.