Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

Hewitt, S.N.; Choi, R.; Kelley, A.; Crowther, G.J.; Napuli, A.J.; Van Voorhis, W.C.

doi:10.1107/S1744309111022159

Acta Crystallographica Section F
Acta Crystallographica
Section F STRUCTURAL BIOLOGY COMMUNICATIONS

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Figure 4
Example of more typical protein-purification products. Large quantities of fused protein running at the molecular weight of target protein and MBP combined are visible in the total (T) and soluble (S) fractions. (a) After cleavage, two forms of the protein remain visible: a band corresponding to the size of MBP and a band corresponding to the size of MBP plus the target protein; very little to no target protein remains in solution. A chromatogram (b) and SDS–PAGE (c) of SEC fractions from uncleaved sample indicate a heterogenous solution of either the recombinant protein expressed with MBP (higher molecular-weight band) or MBP alone (lower molecular-weight band). M, molecular-weight marker; T, total lysate; S, soluble fraction; FT, flowthrough from IMAC after 3C cleavage; W, wash after 3C cleavage; E, eluate with 250 mM imidazole from IMAC after 3C cleavage. The protein expressed and purified was Brucella abortus blue (type 1) copper protein.

STRUCTURAL BIOLOGY
COMMUNICATIONS

ISSN: 2053-230X

Volume 67| Part 9| August 2011| Pages 1006-1009

doi:10.1107/S1744309111022159

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