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Figure 3
SDS–PAGE and SEC chromatogram of an exemplary rescued target which was easily purified. Even after cleavage and removal of the His-MBP tag, the target protein remained soluble and expressed at the expected size. (a) SDS–PAGE of samples from initial IMAC purification and subsequent 3C cleavage of the His-MBP tag. P represents pure sample after the first IMAC step; the observed molecular weight corresponds to the expected size of the recombinant protein expressed with fused MBP. After cleavage with 3C protease, His-MBP is retained on subsequent IMAC (E), while flowthrough (FT) and wash (W) samples contained protein that passes over the nickel column unbound. Unbound recombinant protein was pooled and subjected to SEC. (b) Chromatogram of SEC indicating fractions and sieving properties of smaller molecular-weight protein (without MBP tag). (c) SDS–PAGE of SEC fractions showing the purity of the final preparation. M, molecular-weight marker; T, sample from total lysate; S, sample from soluble fraction after centrifugation. The protein expressed and purified was an uncharacterized protein from Coccidioides immitis.

ISSN: 2053-230X
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