Expression and purification of holo-ACP has been described previously [11 (link)]. Briefly, the acyl-carrier-protein and holo-(acyl-carrier-protein) synthase genes were cloned into a pETDUET vector (EMD-Millipore). Both proteins were expressed simultaneously following the same steps as FabD expression (above). After lysing and centrifuging the cells the clarified lysate was applied to an 8 ml nickel column (Qiagen) and an imidazole gradient was run from 0–500 mM imidazole in buffers containing 20 mM Tris-HCl, 500 mM sodium chloride, pH 8.1. Fractions containing holo-ACP were diluted to reduce sodium chloride to 100 mM and loaded onto a 110 ml Q-Sepharose column. A linear gradient from 200–600 mM potassium chloride was applied to the column and the holo-ACP eluted around 500 mM potassium chloride. Fractions containing ACP were concentrated and exchanged into a buffer containing 25 mM MOPS, 100 mM sodium chloride, 1 mM DTT, pH 7.1 using a Superdex S75 column (GE). Fractions containing holo-ACP were concentrated using a 3 kDa amicon filter unit and stored at −80°C in 10% glycerol. The ACP extinction coefficient used for quantification was 1490 M−1cm−1.
Petduet vector
Manufactured by Merck Group
The PETDUET vector is a genetic engineering tool used for the expression of recombinant proteins. It is designed to facilitate the simultaneous expression of multiple proteins in the same host cell. The PETDUET vector provides a dual-promoter system that allows for the co-expression of two target genes, enabling the study of protein-protein interactions or the production of multi-subunit protein complexes.
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Lab products found in correlation
2 protocols using petduet vector
1
Producing Holo-Acyl Carrier Protein
2
Expression and Purification of Holo-ACP
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Expression and purification of holo-ACP has been described previously [11 (link)]. Briefly, the acyl-carrier-protein and holo-(acyl-carrier-protein) synthase genes were cloned into a pETDUET vector (EMD-Millipore). Both proteins were expressed simultaneously following the same steps as FabD expression (above). After lysing and centrifuging the cells the clarified lysate was applied to an 8 ml nickel column (Qiagen) and an imidazole gradient was run from 0–500 mM imidazole in buffers containing 20 mM Tris-HCl, 500 mM sodium chloride, pH 8.1. Fractions containing holo-ACP were diluted to reduce sodium chloride to 100 mM and loaded onto a 110 ml Q-Sepharose column. A linear gradient from 200–600 mM potassium chloride was applied to the column and the holo-ACP eluted around 500 mM potassium chloride. Fractions containing ACP were concentrated and exchanged into a buffer containing 25 mM MOPS, 100 mM sodium chloride, 1 mM DTT, pH 7.1 using a Superdex S75 column (GE). Fractions containing holo-ACP were concentrated using a 3 kDa amicon filter unit and stored at −80°C in 10% glycerol. The ACP extinction coefficient used for quantification was 1490 M-1cm-1.
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