Protocol

Insoluble Proteins

Volume 1258 of the series Methods in Molecular Biology pp 181-205

Date:

Insect Cells–Baculovirus System for the Production of Difficult to Express Proteins

  • Judit Osz-PapaiAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS
  • , Laura RaduAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS
  • , Wassim AbdulrahmanAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS
  • , Isabelle Kolb-CheynelAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS
  • , Nathalie Troffer-CharlierAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS
  • , Catherine BirckAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS
  • , Arnaud PoterszmanAffiliated withInstitut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS Email author 

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Abstract

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

Key words

Baculovirus Insect cells Homologous recombination Protein expression and production Solubility screen