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. 2010 Feb;69(2):147-52.
doi: 10.1016/j.pep.200908001. Epub 2009 Aug 11.

Expression and purification of his-tagged recombinant mouse zeta-crystallin

Affiliations

Expression and purification of his-tagged recombinant mouse zeta-crystallin

Mukoma F Simpanya et al. Protein Expr Purif. 2010 Feb.

Abstract

Zeta-crystallin is an NADPH-binding protein consisting of four identical 35kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse zeta-crystallin, which was then purified to homogeneity. The yield of pure recombinant mouse zeta-crystallin was five times that obtained previously for purification of recombinant guinea pig zeta-crystallin. The quinone oxidoreductase activity of purified his-tagged recombinant mouse zeta-crystallin was comparable to that of purified native guinea pig lens zeta-crystallin, and to that previously reported for recombinant guinea pig zeta-crystallin. The method permits production of substantial amounts of recombinant zeta-crystallin for conducting studies on the biological role of this interesting protein, which exists in such high concentration in the lenses of certain species.

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Figures

Figure 1
Figure 1
Construction of the pET-15b-ζ vector. The Nde I / Bgl II fragment of the PCR product (containing a his6 tag and the coding region of ζ-crystallin) was inserted into the Nde I and Bgl II sites of the pET-15b vector.
Figure 2
Figure 2
Purification of his-tagged recombinant mouse ζ-crystallin using DEAE Sepharose. ζ-crystallin was eluted with an increasing salt concentration to 1 M NaCl in 10 mM Tris/HCl (pH 7.4), containing 0.5 mM EDTA. The cross-hatched area represents pooled fractions of ζ-crystallin, the presence of which was determined by analysis of each fraction by SDS-PAGE. Pooled fractions were dialyzed and purified further with the use of Ni-NTA column (Figure 3).
Figure 3
Figure 3
Purification of his-tagged recombinant mouse ζ-crystallin on a Nickel-NTA column. Pooled fractions obtained from a DEAE Sepharose column (Figure 2) were applied to a Ni-NTA column, and ζ-crystallin was eluted with buffer containing increasing concentrations of imidazole up to 500 mM. ζ-crystallin eluted in fractions 33–37.
Figure 4
Figure 4
SDS-PAGE and Western blot analysis at various stages of purification of recombinant his-tagged mouse ζ-crystallin using DEAE Sepharose and Nickel-NTA chromatography. A, MW markers. B, Coomassie stain, DEAE Sepharose purification (Figure 2). C, Coomassie stain, Nickel-NTA purification (Figure 3). D, Western blot of lane C using guinea pig ζ-crystallin antibody.
Figure 5
Figure 5
Guinea pig lens ζ-crystallin, purified previously with Blue Sepharose, was purified further on a CM Fast Flow (HiTrap CM FF agarose) column. ζ-crystallin was eluted from the column with an increasing salt concentration of up to 0.5 M NaCl in 10 mM Tris-HCl buffer (pH 7.4), containing 0.5 mM EDTA and 5 mM β-mercaptoethanol. ζ-crystallin eluted between 0 and 50% salt of 0.5 M NaCl (128–133 min elution time).
Figure 6
Figure 6
SDS-PAGE analysis at various stages of purification of native guinea pig lens ζ-crystallin using Blue Sepharose and a CM FF column (Figure 5). A, MW markers. B, Crude guinea pig lens water soluble cortical protein. C, ζ-crystallin after Blue Sepharose purification. D, ζ-crystallin after purification by CM FF column chromatography.
Figure 7
Figure 7
SDS-PAGE analysis of purified his-tagged recombinant mouse and native guinea pig lens ζ-crystallins. A, MW markers. B, Coomassie stain of his-tagged recombinant mouse ζ-crystallin after Ni-NTA purification. C, Coomassie stain of native guinea pig lens ζ-crystallin after CM FF purification.

References

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