An automated in vitro protein folding screen applied to a human dynactin subunit.
The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies). Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives. However, it is difficult to assess the success of such a screen if no biological assay is available. We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy. The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E. coli. The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses.
Scheich
Christoph
C
Proteinstrukturfabrik, Heubnerweg 6, Berlin 14059, Germany. scheich@molgen.mpg.de
Niesen
Frank H
FH
Seckler
Robert
R
Büssow
Konrad
K
D000818:Animals; D001331:Automation; D002942:Circular Dichroism; D000072159:Dynactin Complex; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D017510:Protein Folding; D020673:Protein Renaturation; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013552:Swine; D014364:Tryptophan / Q000737:chemistry
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014364:Tryptophan; D008291:Malate Dehydrogenase