Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC.
Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.
Stemp
Markus J
MJ
Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Guha
Suranjana
S
Hartl
F Ulrich
FU
Barral
José M
JM
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism*; D002457:Cell Extracts; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D056427:t-Complex Genome Region; D000095983:Bacterial Lysates
D000199:Actins; D002457:Cell Extracts; D018835:Chaperonin 10; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins; D003850:Deoxyribonuclease I; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; C030259:Broncho-Vaxom; D000095983:Bacterial Lysates; C494635:DnaJ protein, E coli; C489400:GrpE protein, E coli; C063842:dnaK protein, E coli