Prevention of aggregation after refolding by balanced stabilization-destabilization: production of the Arabidopsis thaliana protein APG8a (At4g21980) for NMR structure determination.
The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process. The protein is an interesting candidate for structure determination by NMR spectroscopy. Toward this end, APG8a fused to an N-terminal His-tag has been expressed in Escherichia coli under a T7 expression system, refolded in vitro, and kept soluble by slight destabilization. The expressed protein appeared in both the soluble and the insoluble fractions. The whole-cell lysate was denatured by the addition of guanidinium chloride. The protein was immobilized on nickel-agarose resin and refolded by stepwise decrement of the denaturant. The elution buffer was 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, 300 mM NaCl, and 1 M imidazole. After the removal of imidazole by ultrafiltration, the His-tag was cleaved with biotinylated thrombin. The protein product was kept in 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, and 300 mM NaCl. The protein was found to aggregate extensively over time if any one of the three ingredients (sodium chloride, urea, or glycerol) was omitted. The yield of the protein was around 20 mg/L Luria-Bertani culture medium. The (1)H-(15)N NMR correlation spectrum of (15)N-labeled APG8a showed the characteristic signature of a folded protein; thus, the solutes appear to have no deleterious effect on the sample. These solution conditions kept the protein soluble and unaggregated for at least 2 days (enough time for NMR data collection). This approach of balanced stabilization-destabilization may offer a general approach for structural investigations of proteins that tend to aggregate.
Chae
Young Kee
YK
Department of Applied Chemistry and Molecular Biology, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Korea.
Im
Hana
H
Zhao
Qin
Q
Doelling
Jed H
JH
Vierstra
Richard D
RD
Markley
John L
JL
D029681:Arabidopsis Proteins; D000071190:Autophagy-Related Protein 8 Family; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C491896:APG8a protein, Arabidopsis; C491895:APG8i protein, Arabidopsis; C113757:ATG8 protein, S cerevisiae; D029681:Arabidopsis Proteins; D000071190:Autophagy-Related Protein 8 Family; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins