High-Throughput Purification of Proteins from Escherichia Coli

Pascal Braun, Joshua LaBaer

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

This chapter describes a procedure for the expression and purification of proteins from Escherichia coli in an automatable 96-well format. The first step in high-throughput expression and purification of proteins is obtaining the corresponding protein expression constructs. The expression of proteins often requires that the gene sequences of interest be placed under the control of a specific transcriptional promoter near specific translational signals in association with a particular selectable marker. The addition of peptide tags requires that the un-translated regions flanking the coding sequences be removed. Finally, the importance of ensuring that the expression clone sequences accurately reflect the natural gene sequence cannot be overemphasized. Even subtle changes in the amino acid sequences of proteins can have profound effects on protein function. One needs to resuspend pellets by shaking the plate on a Beckman Thermoshaker for 30-60 min at 750 rpm at room temperature. This Swizzler improves lyses under both denaturing and non-denaturing conditions. If the goal is to recover functional protein by renaturation, the shaking speed should be reduced to 300 rpm after 5 min to minimize harmful oxidation of proteins.

Original languageEnglish (US)
Title of host publicationCell Biology, Four-Volume Set
PublisherElsevier Inc.
Pages73-77
Number of pages5
Volume4
ISBN (Print)9780121647308
DOIs
StatePublished - 2006
Externally publishedYes

Fingerprint

Escherichia coli Proteins
Purification
Throughput
Proteins
Protein Renaturation
Genes
Amino Acid Sequence
Clone Cells
Association reactions
Peptides
Temperature
Amino Acids
Oxidation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Braun, P., & LaBaer, J. (2006). High-Throughput Purification of Proteins from Escherichia Coli. In Cell Biology, Four-Volume Set (Vol. 4, pp. 73-77). Elsevier Inc.. https://doi.org/10.1016/B978-012164730-8/50196-9

High-Throughput Purification of Proteins from Escherichia Coli. / Braun, Pascal; LaBaer, Joshua.

Cell Biology, Four-Volume Set. Vol. 4 Elsevier Inc., 2006. p. 73-77.

Research output: Chapter in Book/Report/Conference proceedingChapter

Braun, P & LaBaer, J 2006, High-Throughput Purification of Proteins from Escherichia Coli. in Cell Biology, Four-Volume Set. vol. 4, Elsevier Inc., pp. 73-77. https://doi.org/10.1016/B978-012164730-8/50196-9
Braun P, LaBaer J. High-Throughput Purification of Proteins from Escherichia Coli. In Cell Biology, Four-Volume Set. Vol. 4. Elsevier Inc. 2006. p. 73-77 https://doi.org/10.1016/B978-012164730-8/50196-9
Braun, Pascal ; LaBaer, Joshua. / High-Throughput Purification of Proteins from Escherichia Coli. Cell Biology, Four-Volume Set. Vol. 4 Elsevier Inc., 2006. pp. 73-77
@inbook{936370b84345428b90101b9af5dddd84,
title = "High-Throughput Purification of Proteins from Escherichia Coli",
abstract = "This chapter describes a procedure for the expression and purification of proteins from Escherichia coli in an automatable 96-well format. The first step in high-throughput expression and purification of proteins is obtaining the corresponding protein expression constructs. The expression of proteins often requires that the gene sequences of interest be placed under the control of a specific transcriptional promoter near specific translational signals in association with a particular selectable marker. The addition of peptide tags requires that the un-translated regions flanking the coding sequences be removed. Finally, the importance of ensuring that the expression clone sequences accurately reflect the natural gene sequence cannot be overemphasized. Even subtle changes in the amino acid sequences of proteins can have profound effects on protein function. One needs to resuspend pellets by shaking the plate on a Beckman Thermoshaker for 30-60 min at 750 rpm at room temperature. This Swizzler improves lyses under both denaturing and non-denaturing conditions. If the goal is to recover functional protein by renaturation, the shaking speed should be reduced to 300 rpm after 5 min to minimize harmful oxidation of proteins.",
author = "Pascal Braun and Joshua LaBaer",
year = "2006",
doi = "10.1016/B978-012164730-8/50196-9",
language = "English (US)",
isbn = "9780121647308",
volume = "4",
pages = "73--77",
booktitle = "Cell Biology, Four-Volume Set",
publisher = "Elsevier Inc.",

}

TY - CHAP

T1 - High-Throughput Purification of Proteins from Escherichia Coli

AU - Braun, Pascal

AU - LaBaer, Joshua

PY - 2006

Y1 - 2006

N2 - This chapter describes a procedure for the expression and purification of proteins from Escherichia coli in an automatable 96-well format. The first step in high-throughput expression and purification of proteins is obtaining the corresponding protein expression constructs. The expression of proteins often requires that the gene sequences of interest be placed under the control of a specific transcriptional promoter near specific translational signals in association with a particular selectable marker. The addition of peptide tags requires that the un-translated regions flanking the coding sequences be removed. Finally, the importance of ensuring that the expression clone sequences accurately reflect the natural gene sequence cannot be overemphasized. Even subtle changes in the amino acid sequences of proteins can have profound effects on protein function. One needs to resuspend pellets by shaking the plate on a Beckman Thermoshaker for 30-60 min at 750 rpm at room temperature. This Swizzler improves lyses under both denaturing and non-denaturing conditions. If the goal is to recover functional protein by renaturation, the shaking speed should be reduced to 300 rpm after 5 min to minimize harmful oxidation of proteins.

AB - This chapter describes a procedure for the expression and purification of proteins from Escherichia coli in an automatable 96-well format. The first step in high-throughput expression and purification of proteins is obtaining the corresponding protein expression constructs. The expression of proteins often requires that the gene sequences of interest be placed under the control of a specific transcriptional promoter near specific translational signals in association with a particular selectable marker. The addition of peptide tags requires that the un-translated regions flanking the coding sequences be removed. Finally, the importance of ensuring that the expression clone sequences accurately reflect the natural gene sequence cannot be overemphasized. Even subtle changes in the amino acid sequences of proteins can have profound effects on protein function. One needs to resuspend pellets by shaking the plate on a Beckman Thermoshaker for 30-60 min at 750 rpm at room temperature. This Swizzler improves lyses under both denaturing and non-denaturing conditions. If the goal is to recover functional protein by renaturation, the shaking speed should be reduced to 300 rpm after 5 min to minimize harmful oxidation of proteins.

UR - http://www.scopus.com/inward/record.url?scp=84881715121&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84881715121&partnerID=8YFLogxK

U2 - 10.1016/B978-012164730-8/50196-9

DO - 10.1016/B978-012164730-8/50196-9

M3 - Chapter

AN - SCOPUS:84881715121

SN - 9780121647308

VL - 4

SP - 73

EP - 77

BT - Cell Biology, Four-Volume Set

PB - Elsevier Inc.

ER -