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Improvements in large-scale production of Tobacco Etch Virus protease

  1. Author:
    Messing,Simon
    Barnhart,Kirsten
    Drew,Matt
    Granato-Guerrero,Natalie
    Grose,Carissa
    Higgins,Brianna
    Hong,Min
    Hull,Jenna
    Perkins,Shelley
    Poon,Ivy
    Ramakrishnan,Nitya
    Seabolt,Amanda
    Taylor,Troy
    Wall,Vanessa
    Wright,Nick
    Gillette,Bill
    Esposito,Dom

  2. Author Address

    Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA. Electronic address: simon.messing@nih.gov.,
    1. Year: 2024
    2. Date: Dec 14
    3. Epub Date: 2024 12 14
  2. Journal: Protein Expression and Purification
    1. Pages: 106648
  4. Type of Article: Article
  5. Article Number: 106648
  1. Abstract:

    Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms. During our work we detected post-translational modification (gluconoylation and phosphogluconoylation) of TEV protease and the subsequent effects this has on the purity of the protein. Subsequently we made our pgl plus bacteria that negates these modifications and their effects. We also introduce a GFP-based assay for measurement of activity and ultimately a new set of protocols for producing 400-500 mg/L TEV protease. Copyright © 2024. Published by Elsevier Inc.

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External Sources

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  2. DOI: 10.1016/j.pep.2024.106648
  3. PMID: 39681152
  4. PII : S1046-5928(24)00220-1

Library Notes

  1. Fiscal Year: FY2024-2025
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