Methods and reagents: Purification of His-Tag fusion proteins from Escherichia coli Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a contaminating protein from Escherichia coli found when histidine-tagged fusion proteins are purified by metal chelate affinity chromatography. For details on how to partake in the newsgroup, see the accompanying box. Fusion proteins containing a peptide tag make it a snap to purify proteins of interest from both eukaryotic and prokaryotic organisms for structural studies, epitope mapping or for the large-scale purification of immunogenic peptides used as vaccines. By cloning a segment of DNA within a plasmid vector with a strongly regulated promoter and several restriction enzyme sites within a polylinker positioned at either end of the translated protein, the gene of interest is fused in frame with a string of nucleotides encoding six histidine residues (His6-Tag). The affinity of the His-Tag for Cu2+, Ni2+, Co2+ or Zn2+ allows the fusion product to be quickly separated from the bulk of other bacterial proteins with up to 95% purity using metal chelate affinity chromatography.[1] The protein is purified by passing an extract of E. coli over-expressing the protein through nickel-agarose or a special metal-containing resin column. The protein of interest binds to the matrix through interactions with the metal ions and can be eluted with a solution of ammonium chloride, glycine, histidine, imidizole, EDTA or high levels of competing metal ions. [2] Several plasmid vectors are now commercially available for the construction of histidine-tagged (His-Tag) fusion proteins, and some kits come complete with all the components necessary for the entire operation, from ligation of the insert into a series of vectors with different reading frames to the purification of the fusion protein using specially designed affinity columns containing a metal-chelating resin. Alternatively, vectors comparable to those obtained from biotechnology companies are available - some for little or no cost [3-5] - but an appropriate binding column would still have to be constructed or purchased in order to purify the expressed proteins. Playing His-Tag *************** One netter, Eric Anderson (anderson@pharmdec.wustl.edu) was using the Xpress [TM] system from Invitrogen (San Jose, CA, USA) with vectors pTrcHis A, B, and C in order to purify a protein from E. coli. Having invested time and effort designing the experiment and cloning his gene into pTrcHis, he experienced some difficulty in the purification step when using Ni-NTA (nickel-nitriloacetic acid) [6] spin columns (QIAGEN Inc., Chatsworth, CA, USA): the eluate appeared to contain a contaminant after he had attempted to recover his His-Tag protein with increasing concentrations of imidizole. Two extra protein bands of approximately 45 kDa and 50 kDa were seen migrating in a sodium dodecyl sulfate (SDS)-polyacrylamide gel when elution was carried out with 25-100 mM imidizole. Since he suspected the extra proteins might be coming from the host E. coli strain, he posted to methds-reagnts asking for some `net-wisdom'. One person suggested that the extra bands could be degradation products if the protein of interest was expected to be larger than those seen migrating in the gel, and that using a cocktail of protease inhibitors could solve the problem. In addition, genes from organisms other than E. coli might possess rare codons that could cause stalling of the ribosome and premature termination of translation, which would produce smaller peptides than that of the fusion product. This was entirely possible since the gene cloned in this case was from Caenorhabdtitis elegans and the expected size of the fusion product (‾65 kDa) was larger. However, it seemed unlikely that the proteins would be degraded because the bacterial cell lysates had been prepared in buffer containing protease inhibitors: 200 uM Pefabloc [TM] (Boehringer Mannheim, Indianapolis, IN, USA), 1 mM pepstatin, 1 mM leupeptin and 5 mM 2-mercaptoethanol. It was also suggested that if the bands were contaminating proteins from the host, a control experiment using the same host stain transformed with an empty vector should reveal any E. coli proteins that bound to the column. When the control experiments were performed, the extra bands still appeared, pinning down the source as the E. coli strain used. Others had already come across a histidine-rich protein from E. coli that sometimes interferes with the His-Tag method by co-purifying or outcompeting recombinant His6-tagged proteins when they are purified using a nickel-agarose column. An E. coli protein consisting of a domain homologous to FK506-binding proteins (FKBPs) has been detected as a persistent contaminant in immobilized metal affinity chromatography of recombinant proteins that have been expressed in E. coli. [7] Owing to a high number of histidine residues (20 out of 196 amino acids), the protein binds tightly to Ni2+ and Zn2+, less tightly to Cu2+ and Co2+, and even less tightly to Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+. Interestingly, the E. coli gene for this histidine-rich protein (GenBank Accession No. Z21496) encodes a protein of nearly 21 kDa, making it unlikely that this was the protein contaminating the column, unless the proteins could somehow form multimers - perhaps by sharing metal ions. One study showed not only that shortened proteins are observed in metal affinity column eluates, but that co-aggregates of proteins could also occur, even in the presence of SDS. Some of these were further revealed to be multimers of the fusion product, which had formed through disulfide bridges between cysteine residues, or co-aggregates of other proteins attached to the fusion product through disulfide bonds. The proteins forming the aggregates could be resolved by adding 20 mM 2-mercaptoethanol to the elution buffer. [3] It is therefore not known whether the contaminant from the column was in fact the His-rich protein, or even whether that protein can be isolated from E. coli Top10 F', the host strain supplied with the kit. In addition to the His-rich protein, researchers have discovered at least three other E. coli proteins that might contaminate their columns, the most abundant of which is a superoxide dismutase. [3] It is reasonable to suppose that the contaminant could be any one of a number of metal-binding proteins. Because they did not interfere with the detection of the expected fusion product by western blot, or the enzyme assay used to confirm the expressed protein, the extra protein bands were ignored. Had they played a more critical role in these experiments, the purification scheme might have been abandoned and chalked up as a waste of time and money. Obviously, for future His-Tag fusion work, it would be a tremendously advantageous to develop a deletion derivative of E. coli that lacks any dispensable gene known to encode proteins that might bind to and contaminate metal ion columns. References [1] Hochuli, E. et al. (1988) Bio/Technology 6, 1321-1325 [2] Kagedal, L. (1989) in Protein Purification: Principles, High Resolution Methods, and Applications (Janson, J.-C. and Ryden, L., eds), pp. 227-251, VCH [3] Stuber, D., Matile, H. and Garotta, G. (1990) in Immunological Methods (Lefkovits, I. and Pernis, B., eds) pp. 121-152, Academic Press [4] Chen, B. P. C. and Hai, T. (1994) Gene 139, 73-75 [5] Baier, G. et al. (1994) BioTechniques 17, 94-99 [6] Hochuli, E., Dobeli, H. and Schacher, A. (1987) J. Chromatogr. 411, 177-184 [7] Wulfing, C., Lombardero, J. and Pluckthun, A. (1994) J. Biol. Chem. 269, 2895-2901 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jul95.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1995. Methods and reagents - Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences 20(7):285-286. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************