Article

Journal of Structural and Functional Genomics

, Volume 6, Issue 1, pp 13-20

First online:

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system

  • Nathan P. CowiesonAffiliated withInstitute for Molecular Bioscience
  • , Pawel ListwanAffiliated withSchool of Molecular and Microbial SciencesCooperative Research Centre for Chronic Inflammatory DiseasesARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience Precinct, University of Queensland
  • , Mareike KurzAffiliated withInstitute for Molecular Bioscience
  • , Anna AagaardAffiliated withInstitute for Molecular Bioscience
  • , Timothy RavasiAffiliated withInstitute for Molecular BioscienceCooperative Research Centre for Chronic Inflammatory DiseasesARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience Precinct, University of Queensland
  • , Christine WellsAffiliated withInstitute for Molecular BioscienceCooperative Research Centre for Chronic Inflammatory DiseasesARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience Precinct, University of Queensland
  • , Thomas HuberAffiliated withDepartment of Mathematics
  • , David A. HumeAffiliated withInstitute for Molecular BioscienceCooperative Research Centre for Chronic Inflammatory DiseasesARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience Precinct, University of Queensland
  • , Bostjan KobeAffiliated withInstitute for Molecular BioscienceSchool of Molecular and Microbial SciencesARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience Precinct, University of Queensland
    • , Jennifer L. MartinAffiliated withInstitute for Molecular BioscienceSchool of Molecular and Microbial SciencesARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience Precinct, University of Queensland Email author 

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Abstract

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.

Keywords

bacterial expression mammalian proteins medium-throughput protein expression screening recombinatorial cloning