Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

T. V S Murthy, Weilin Wu, Q. Q. Qiu, Zhenwei Shi, Joshua LaBaer, Leonardo Brizuela

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500ng after a single affinity purification step from 50μl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.

Original languageEnglish (US)
Pages (from-to)217-225
Number of pages9
JournalProtein Expression and Purification
Volume36
Issue number2
DOIs
StatePublished - Aug 2004
Externally publishedYes

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Cell-Free System
Escherichia coli
Throughput
Proteins
Purification
Bacterial Proteins
Solubility
Pseudomonas aeruginosa
Polyacrylamide Gel Electrophoresis
Clone Cells
Screening
In Vitro Techniques

Keywords

  • High-throughput protein expression
  • In vitro protein synthesis
  • Pseudomonas aeruginosa
  • Two-component system

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems. / Murthy, T. V S; Wu, Weilin; Qiu, Q. Q.; Shi, Zhenwei; LaBaer, Joshua; Brizuela, Leonardo.

In: Protein Expression and Purification, Vol. 36, No. 2, 08.2004, p. 217-225.

Research output: Contribution to journalArticle

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