Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag.

Protein Expression and Purification
S Hesam ShahravanJumi A Shin

Abstract

In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of...Continue Reading

References

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Citations

Feb 15, 2011·Biotechnology Letters·Wen-Hui K Kuo, Howard A Chase
Nov 28, 2013·Protein Expression and Purification·Jane T MooneyMilton T W Hearn
Nov 19, 2010·Protein Engineering, Design & Selection : PEDS·Peter SimeonovThole Zuchner
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Apr 15, 2014·Protein Expression and Purification·Yufei FengDonglai Wu
Sep 6, 2011·Journal of Biotechnology·Alessandra StefanAlejandro Hochkoeppler
Feb 26, 2013·Protein Science : a Publication of the Protein Society·Eliot T Smith, David A Johnson
Mar 3, 2015·Journal of Molecular Recognition : JMR·Jane T MooneyMilton T W Hearn
Apr 26, 2011·Protein Expression and Purification·Marine E GasparianMikhail P Kirpichnikov
Sep 12, 2014·Bioscience, Biotechnology, and Biochemistry·Jing HuiFeng-qing Hu
May 19, 2017·Langmuir : the ACS Journal of Surfaces and Colloids·Shrikrishnan SankaranPascal Jonkheijm
May 21, 2020·Proteins·Christoph ÖhlknechtChris Oostenbrink
Jan 17, 2019·BioMed Research International·María de J López-LópezXianwu Guo
May 11, 2020·Protein Expression and Purification·Jordan BrungardtHarold N Trick
Sep 6, 2020·General and Comparative Endocrinology·Ryuya HasegawaMunetaka Shimizu
Jun 7, 2020·Current Protein & Peptide Science·Vibhor Mishra
May 27, 2021·Bioprocess and Biosystems Engineering·Jiong HuJun Fan

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