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High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 prote...

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Autores principales: Bryan, Cassie M., Bhandari, Janhavi, Napuli, Alberto J., Leibly, David J., Choi, Ryan, Kelley, Angela, Van Voorhis, Wesley C., Edwards, Thomas E., Stewart, Lance J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169394/
https://www.ncbi.nlm.nih.gov/pubmed/21904042
http://dx.doi.org/10.1107/S1744309111018367
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author Bryan, Cassie M.
Bhandari, Janhavi
Napuli, Alberto J.
Leibly, David J.
Choi, Ryan
Kelley, Angela
Van Voorhis, Wesley C.
Edwards, Thomas E.
Stewart, Lance J.
author_facet Bryan, Cassie M.
Bhandari, Janhavi
Napuli, Alberto J.
Leibly, David J.
Choi, Ryan
Kelley, Angela
Van Voorhis, Wesley C.
Edwards, Thomas E.
Stewart, Lance J.
author_sort Bryan, Cassie M.
collection PubMed
description The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.
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spelling pubmed-31693942011-09-21 High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease Bryan, Cassie M. Bhandari, Janhavi Napuli, Alberto J. Leibly, David J. Choi, Ryan Kelley, Angela Van Voorhis, Wesley C. Edwards, Thomas E. Stewart, Lance J. Acta Crystallogr Sect F Struct Biol Cryst Commun Laboratory Communications The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography. International Union of Crystallography 2011-08-13 /pmc/articles/PMC3169394/ /pubmed/21904042 http://dx.doi.org/10.1107/S1744309111018367 Text en © Bryan et al. 2011 http://creativecommons.org/licenses/by/2.0/uk/ This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
spellingShingle Laboratory Communications
Bryan, Cassie M.
Bhandari, Janhavi
Napuli, Alberto J.
Leibly, David J.
Choi, Ryan
Kelley, Angela
Van Voorhis, Wesley C.
Edwards, Thomas E.
Stewart, Lance J.
High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease
title High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease
title_full High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease
title_fullStr High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease
title_full_unstemmed High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease
title_short High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease
title_sort high-throughput protein production and purification at the seattle structural genomics center for infectious disease
topic Laboratory Communications
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169394/
https://www.ncbi.nlm.nih.gov/pubmed/21904042
http://dx.doi.org/10.1107/S1744309111018367
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