Screening methods to determine biophysical properties of proteins in structural genomics.
We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy. The method constitutes an extension of previously described protocols for gene expression and protein solubility screening [M. Hammarström et al., (2002), Protein Science 11, 313]. Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day. The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner. Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations. The purification method can be automated, scaled up or down, and extended to a traditional purification scheme. We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.
Woestenenk
Esmeralda A
EA
Department of Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
Hammarström
Martin
M
Härd
Torleif
T
Berglund
Helena
H
D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D023281:Genomics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D011506:Proteins