The gene encoding EstA was amplified by PCR, without the predicted signal peptide (the first 16 amino acids) and without its stop codon (fused to a His6 tag), using chromosomal DNA of T. maritima as a template and the two primers BG1962 (5′-GCGCCATGGAGGATGTTACTGTGAAGAGTG-3′) and BG1963 (5′-GCGCTCGAGTCTACTTTGTTCAAACAGCCAC-3′), introducing NcoI and XhoI restriction sites. The generated PCR product was digested by NcoI and XhoI and the product was purified and ligated into pET-24d digested with the same restriction enzymes, resulting in the plasmid pWUR350. The construct was designed with a hexahistidine tag engineered at the C-terminus of the enzyme to facilitate purification. E. coli BL21(DE3)/pSJS1244 was transformed with pWUR350.
A single colony was used to inoculate 4 ml Luria–Bertani medium containing kanamycin and spectinomycin (both 50 µg ml−1) and incubated overnight at 310 K with shaking. The preculture was used to inoculate (1:1000) 2 l of the same medium and growth was continued for 8 h (an OD600 above 2.0 was reached). Subsequently, the culture was induced by adding IPTG (isopropyl β-d-1-thiogalactopyranoside) to a final concentration of 0.5 mM. The culture was incubated for a further 16 h at 310 K.
Cells were harvested by centrifugation at 10 000g
for 10 min. The cell pellet was resuspended in 30 ml buffer (50 mM
Tris–HCl pH 7.5, 300 mM
NaCl, 10 mM
imidazole). The cells were disrupted by passage twice through a French press at 110 MPa. The crude cell extract was treated with DNAse I at room temperature for 30 min and subsequently centrifuged at 43 000g
for 30 min in order to remove cell debris. The supernatant was heated at 343 K for 30 min and then centrifuged to remove the precipitated proteins. The supernatant was filtered and applied onto a nickel-chelating column packed with 20 ml Ni–NTA His-Bind Resin (Novagen) and equilibrated in 50 mM
Tris–HCl buffer pH 7.8 containing 300 mM
NaCl. The column was washed with 20 mM
imidazole in the same buffer and proteins were subsequently eluted with a linear gradient of 20–500 mM
imidazole in 50 mM
Tris–HCl pH 7.5, 300 mM
NaCl. Fractions containing esterase activity were pooled and applied onto a Hi-Prep desalting column (Amersham Biosciences) equilibrated with 50 mM
disodium phosphate buffer pH 7.5. The homogeneity of the protein was checked by SDS–PAGE and activity staining of the SDS–PAGE gels using α-naphtyl acetate, as described previously (Levisson et al.
). The protein concentration was determined at 280 nm using a NanoDrop ND-1000 Spectrophotometer (NanoDrop). For the preparation of selenomethionine-substituted EstA (SeMet-EstA), the overproducing strain was grown as described previously (Akerboom et al.
) and the enzyme was purified as described above. EstA and SeMet-EstA were assayed for esterase activity using p
-nitrophenyl-valerate as a substrate (data not shown).