Overexpression
plasmids were constructed by subcloning TEM-1 into a pET24 vector
(Life Technologies) with its native export signal sequence replaced
by the OmpA signal sequence to maximize export efficiency.21 (link) Variants were constructed via site-directed
mutagenesis and verified by DNA sequencing. Plasmids were transformed
into BL21(DE3) cells (Life Technologies) for expression under a T7
promoter control. Cells were induced with 1 mM IPTG at OD = 0.6 and
grown at 18 °C for 15 h before harvesting.
TEM β-lactamases
were isolated from the periplasmic fraction using osmotic shock lysis:
Cells were resuspended in 30 mM Tris-Cl, pH 8, and 20% sucrose and
stirred for 10 min at room temperature. After centrifugation, the
pellet was resuspended in ice-cold 5 mM MgSO4 and stirred
for 10 min at 4 °C. After centrifugation, the supernatant was
dialyzed against 20 mM sodium acetate, pH 5.5, and purified using
cation exchange chromatography (HiTrap Capto S column, GE Healthcare).
All variants eluted between 10 and 20% NaCl. Proteins were concentrated,
dialyzed in storage buffer (20 mM Tris, pH 8.0), and final concentrations
were measured in Edelhoch buffer using calculated extinction coefficients
based on the number of tryptophans, tyrosines, and disulfide bonds.22 (link)
plasmids were constructed by subcloning TEM-1 into a pET24 vector
(Life Technologies) with its native export signal sequence replaced
by the OmpA signal sequence to maximize export efficiency.21 (link) Variants were constructed via site-directed
mutagenesis and verified by DNA sequencing. Plasmids were transformed
into BL21(DE3) cells (Life Technologies) for expression under a T7
promoter control. Cells were induced with 1 mM IPTG at OD = 0.6 and
grown at 18 °C for 15 h before harvesting.
TEM β-lactamases
were isolated from the periplasmic fraction using osmotic shock lysis:
Cells were resuspended in 30 mM Tris-Cl, pH 8, and 20% sucrose and
stirred for 10 min at room temperature. After centrifugation, the
pellet was resuspended in ice-cold 5 mM MgSO4 and stirred
for 10 min at 4 °C. After centrifugation, the supernatant was
dialyzed against 20 mM sodium acetate, pH 5.5, and purified using
cation exchange chromatography (HiTrap Capto S column, GE Healthcare).
All variants eluted between 10 and 20% NaCl. Proteins were concentrated,
dialyzed in storage buffer (20 mM Tris, pH 8.0), and final concentrations
were measured in Edelhoch buffer using calculated extinction coefficients
based on the number of tryptophans, tyrosines, and disulfide bonds.22 (link)