Hexahistidine [His6]-tag dependent protein dimerization: a cautionary tale

• Autorzy: Wu J. , Filutowicz M.
• Języki publikacji: EN

Abstrakty

EN

Nickel nitrilotriacetic acid (Ni2+-NTA) immobilization of hexahistidine (His6) tagged proteins has become one of the most commonly used methods of affinity chromatography. Perhaps the greatest utility of this protein purification method stems from the general belief that His-tagged proteins (comprised of His6) are little affected in their activities or efficiencies, while alterations in specificity are unexpected. Although this is certainly true in many instances, we present a case in which the biochemical properties of proteins being studied were fundamentally altered due to the presence of His-tags. We carried out these studies using variants of the π30.5 protein of plasmid R6K, a DNA binding protein which negatively regulates plasmid replication. π30.5 can bind DNA containing a target sequence (TGAGR) arranged either asymmetrically (direct repeats) in the g origin, or symmetrically in inverted half-repeats (IR˘s) in the operator of its own gene, pir. Importantly, dimers of p protein bind to an IR; this property allows researchers to quickly assess whether different regulatory variants of p proteins exhibit altered dimerization properties. For example, π30.5 containing a single amino acid substitution, F107S (π20030.5), has been shown to be monomeric in solution and dimers were not observed bound to IR˘s. Here we demonstrate that the presence of a His-tag partially restores the ability of π20030.5 to dimerize in solution and bind to an IR in dimeric form. This report sends an important message that (other) proteins containing His-tags may differ from their wild type counterparts in dimerization/oligomerization properties.

Słowa kluczowe

EN

affinity chromatography; replication initiator; nickel nitrilotriacetic acid; DNA binding; hexahistidine; protein purification method; protein dimerization

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 1999
• Tom: 46
• Numer: 3
• Opis fizyczny: p.591-599,fig.

Twórcy

autor

Wu J.

• University of Wisconsin, Madison, WI 53706, USA

autor

Filutowicz M.

Bibliografia

• EUenberger, T.E., Brandl, C.J., Struhl, K. & Harri­son, S.C. (1992) The GCN4 basic region LZ binds DNA as a dimer of uninterrupted al­pha-helices: Crystal structure of the pro­tein-DNA complex. Cell 71, 1223-1237.
• Filutowicz, M., Davis, G., Greener, A. & Helinski, D. (1985) Autorepressor properties of the ^-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 13, 103-114.
• Filutowicz, M., Dellis, S., Levchenko, I., Urh, M., Wu, F. & York, D. (1994) Regulation of replica­tion of an iteron-containing DNA molecule. Prog. NucL Acid Res. MoL Biol 48, 239-273.
• Filutowicz, M. & Rakowski, S.A. (1998) Regula­tory implications of protein assemblies at the y origin of plasmid R6K — a review. Gene 223, 195-204.
• Garcia de Viedma, D., Giraldo, R., Rivas, G., Fernandez-Tresguerres. E. & Diaz-Orejas. R. (1996) A leucine zipper motif determines dif­ferent functions in a DNA replication protein. EMBOJ. 15,925-934.
• Glover, J.N. & Harrison, S.C. (1995) Crystal struc­ture of the heterodimeric bZIP transcription factor c-Fos-cJun bound to DNA. Nature 373, 257-261.
• Greener, A., Filutowicz, M., McEachern, M.J. & Helinski, D.R. (1990) N-terminal truncated forms of the Afunctional ji initiation protein express negative activity on plasmid R6K rep­lication. MoL Gen. Genet. 224, 24-32.
• Hope, I .A. & Struhl, K. (1987) GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EAfBO J. 6,2781-2784.
• Inuzuka, M. & Helinski, D.R. (1978) Replication of antibiotic resistance plasmid R6K DNA in vi­tro. Biochemistry 17, 2567-2573.
• Kelley, W.L. & Bastia, D. (1985) Replication initia­tor protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc. Natl Acad. Sci. U.S.A. 82, 2574-2578.
• Kolter, R., Inuzuka, M. & Helinski, D.R. (1978) 7Va/w-complementation-
• Levchenko, I., York, D. & Filutowicz, M. (1994) The dimerization domain of R6K plasmid rep­lication initiator protein n revealed by analy­sis of a truncated protein. Gene 145,65-68.
• Lu, Y., Datta, H. & Bastia, D. (1998) Mechanistic studies of initiator-initiator interaction and replication initiation. EMBO J. 17, 5192- 5200.
• O'Shea, E.K., Klemm, J.D., Kim, P.S. & Alber, T. (1991) X-ray structure of the GCN4 leucine zip­per, a two-strand, parallel coiled coil. Science 254,539-544.
• O'Shea, E.K., Rutkowski, R. & Kim, P.S. (1989) Ev­idence that the leucine zipper is a coiled coil. Science 243, 538-542.
• Ratnakar, P.V.A.L., Mohanty, B.K., Lobert, M. & Bastia, D. (1996) The replication initiator pro­tein n of the plasmid R6K specifically inter­acts with the host-encoded helicase DnaB. Proc. Nati Acad. Sci. U.S.A. 93,5522-5526.
• Saudek, U., Pastore, A., Castiglione-Morelli, M.A., Frank, R., Gansepohl, H. & Gibson, T. (1991) The solution structure of a leucine zipper mo­tif peptide. Protein Eng. 4, 519-529.
• Shafferman, A., Kolter, R., Stalker, D.M. & Helinski, D.R. (1982) Plasmid R6K replica­tion. III. Regulatory properties of the Jt initia­tion protein. J. MoL BioL 161, 57-76.
• Stalker, D.M., Kolter, R. & Helinski, D.R. (1982) Plasmid R6K DNA replication. I. Complete nu­cleotide sequence of an autonomously replicat­ing segment. J. MoL BioL 161, 33-43.
• Urh, M., Wu, J., Wu, J.W., Forest, K., Inman, R.B. & Filutowicz, M. (1998) Assemblies of replica­tion initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces. J. Mol Biol 283, 619-631.
• Wu, J., Sektas, M., Chen, D. & Filutowicz, M. (1997) Two forms of replication initiator pro­tein: Positive and negative controls. Proc. Natl. Acad. Sci. U.S.A. 94, 13967-13972.
• York, D. & Filutowicz, M. (1993) Autor- egulation-deficient mutant of plasmid R6K-encoded 7i protein distinguishes between palindromic and nonpalindromic binding sites. J. Biol Chem. 268, 21854-21861.
• York, D., Ivanov, V., Gan, J. & Filutowicz, M. (1992) Translational options for the pir gene of plasmid R6K: Multiple forms of the replication initiator protein tc. Gene 116,7-12.