6xHis-Ni-NTA Chromatography as a Superior Technique in Recombinant Protein Expressiod/Purification

Crowe, Joanne; Döbeli, Heinz; Gentz, Reiner; Hochuli, Erich; Stüber, Dietrich; Henco, Karsten

doi:10.1385/0-89603-258-2:371

Cited by 183 publications

(183 citation statements)

References 7 publications

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“…Thus, the affinity tag usually does not need to be removed following protein purification. 12 If necessary, the affinity tag can be removed by use of a protease cleavage site inserted between the tag and the protein. 13 Polyhistidine affinity tags are small enough to be incorporated easily into any expression vector.…”

Section: Incorporation Of the Polyhistidine Affinity Tagmentioning

confidence: 99%

“…Metal leaching from the matrix during purification causes lowered yields and impure products. 12 More recently, purification of polyhistidine affinity-tagged proteins has been facilitated by the development of the commercially available matrices nickel-nitrilotriacetic acid (Ni 2+ -NTA) 17 and Co 2+ -carboxylmethylaspartate (Co 2+ -CMA), 18 which are coupled to a solid support resin. These matrices securely coordinate metal ions through four coordination sites while leaving two of the transition metal coordination sites exposed to interact with histidine residues in the affinity tag.…”

Section: Affinity Matricesmentioning

confidence: 99%

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[16] Purification of proteins using polyhistidine affinity tags

Bornhorst

Falke²

2000

Methods in Enzymology

456

290

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The expression and subsequent purification of recombinant proteins are widely employed in biochemical studies. A powerful purification method involves the use of peptide affinity tags, which are fused to the protein of interest and used to expedite protein purification via affinity chromatography. 1,2 A widely employed method utilizes immobilized metal-affinity chromatography (IMAC) to purify recombinant proteins containing a short affinity tag consisting of polyhistidine residues. IMAC is based on the interactions between a transition metal ion (Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ ) immobilized on a matrix and specific amino acid side chains. Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal. Peptides containing sequences of consecutive histidine residues are efficiently retained on IMAC column matrices. Following washing of the matrix material, peptides containing polyhistidine sequences can be easily eluted by either adjusting the pH of the column buffer or adding free imidazole to the column buffer. 3

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Section: Incorporation Of the Polyhistidine Affinity Tagmentioning

confidence: 99%

Section: Affinity Matricesmentioning

confidence: 99%

[16] Purification of proteins using polyhistidine affinity tags

Bornhorst

Falke²

2000

Methods in Enzymology

456

290

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“…Both recombinant proteins accumulated in cytoplasmic inclusion bodies (IBs), which were purified as described (7) but without preparation of spheroplasts prior to IB preparation. The IBs were solubilized in 6 M GuHCl/0.1 M sodium phosphate/0.01 M Tris-HCl, pH 8.0/5 mM 2-mercaptoethanol, and 10 mg was loaded onto a Ni-NTA column (Qiagen) (8). The column was washed with 50 ml of wash buffer (8 M (9).…”

mentioning

confidence: 99%

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Scherf

Pastan

Willingham

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

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“…A similar approach has been used before to purify proteins from mycobacteria successfully, without the histidine tag compromising the function or immunogenicity of the protein (Crowe et al, 1994;Triccas et al, 1998).…”

Section: Purification Of the Histidine-tagged Hybrid-1 Fusion Proteinmentioning

confidence: 99%

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

Tsolaki

Nagy

Leiva

et al. 2013

Molecular Immunology

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In this study, we investigated the potential molecular and immunological differences resulting from production of the recombinant fusion proteins (Hybrid-1, comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis) in two different expression systems, namely M. smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the antigen. We investigated the immunogenicity from both from E. coli-derived Hybrid-1 fusion and M.smegmatis-derived Hybrid-1 fusion in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trails, as it provided protein of excellent yield, stability and free from lipopolysaccharide.3

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6xHis-Ni-NTA Chromatography as a Superior Technique in Recombinant Protein Expressiod/Purification

Cited by 183 publications

References 7 publications

[16] Purification of proteins using polyhistidine affinity tags

[16] Purification of proteins using polyhistidine affinity tags

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

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