Biochemical Methods

2.4.1 Gene Expression and Protein Purification

2.4.1.1. Expression and Purification of KTq wild-type and variants

PGDR11 expression vector harbouring the respective KlenTaq gene was transformed into E. coli BL21 (DE3). For expression, a 5 mL over-night culture of these cells was used to inoculate 500 mL LB media supplemented with 100 mg/L carbenicillin. Cells were grown at 37 °C at 200 rpm shaking to an OD of 0.6-0.8. Expression was induced by addition of IPTG to a final concentration of 1 mM. After 4 h expression, cells were harvested by centrifugation (4 °C, 4000 x g, 30 min) and stored over-night at -20 °C. Cells were lysed in 20 mL 1x KlenTaq Lysis Buffer supplemented with 1 mM Benzamidine at 37 °C for 15 min, followed by heat denaturation of host proteins at 75 °C for 45 min. Ultra-centrifugation (Ultracentrifuge, L-60) at 40 000 rpm for 60 min at 4 °C was used to remove bacterial cell debris.

Supernatants were incubated with 50 % Ni-IDA sepharose slurry in the presence of 5 mM imidazole for 1.5 h at 4 °C gently shaking. After washing with 30 mL 1x KlenTaq Washing Buffer containing 20 mM imidazol, elution was carried out using 6 mL 1x KlenTaq Elution Buffer. The imidazole was removed using VivaSpin columns and 1x KlenTaq Elution Buffer without imidazole. For storage, glycerol was added to a final concentration of 50 %, (NH4)2SO4

to a final concentration of 16 mM, Tween20 to a final concentration of 0.1 %, Tris·HCl pH 9.2 to a final concentration of 50 mM and MgCl2 to a final concentration of 2.5 mM (1x KlenTaq Storage Buffer). Purified enzymes were stored at -20 °C. Concentration was determined by Bradford Assay or SDS-PAGE analysis.

2.4.1.2. Expression and Purification of KTq wild-type and RT-KTq 2 for crystallization

PGDR11 expression vector harbouring the respective KlenTaq gene (wild-type, RT-KTq 2 (codon optimized) without His-tag in pGDR11*, chapter VII 2.3.4.) was transformed into concentration of 1 mM. After 4.5 h expression, cells were harvested by centrifugation (4 °C, 4000 x g, 30 min) and stored over-night at -20 °C. Cells were lysed in 20 mL 1x KlenTaq Lysis Buffer II containing 0.7 mg/mL lysozyme at room temperature for 60 min, followed by heat denaturation of host proteins at 75 °C for 45 min. Ultra-centrifugation (Ultracentrifuge, L-60) at 40 000 rpm for 60 min at 4 °C was used to remove bacterial cell debris.

Afterwards, supernatants were treated stepwise with 5 % polyethylenimine (PEI) to remove bacterial DNA in the supernatant. After PEI was added, the suspension was shaken for 30 min at 4 °C. Then, centrifugation (4000 x g, 30 min, 4 °C) was carried out before adding additional PEI. These steps were repeated three times. The supernatant was then filtrated using a syringe sterile filter (0.45 µm) and afterwards loaded onto a Q sepharose column (kindly provided by Karin Betz). The protein was purified via anion exchange chromatography on an ÄKTApurifier. The column was equilibrated with 2 column volumes (CV) of 1x KlenTaq Ion Exchange Buffer I, followed by 2 CV of 1x KlenTaq Ion Exchange Buffer II and again 2 CV of buffer I. Elution was carried out at 4 °C by applying a gradient of 0‐1 M NaCl (0‐50 % buffer II, pH 8.55) with a flow rate of 1 mL/min.

In detail:

150 mL 100 % buffer I (Washing step) 250 mL 0-8 % buffer II (Elution) 100 mL 8-20 % buffer II (Elution)

Fractions were analysed by SDS-PAGE and pooled. Protein was stored at 4 °C. Expression and purification yielded 26 mg pure protein of KTq wild-type and 10 mg of RT-KTq 2 from 1 L expression culture.

2.4.1.3. Expression and Purification of Taq wild-type and variants

PGDR11 expression vector harbouring the respective Taq gene was transformed into E. coli BL21 (DE3). For expression, an over-night culture (5 mL each) of these cells was used to inoculate 4 x 500 mL LB media supplemented with 100 mg/L carbenicillin. Cells were grown at 37 °C at 200 rpm shaking to an OD of 0.6-0.8. Expression was induced by addition of IPTG to a final concentration of 0.5 mM. After 4.5 h expression, cells were harvested by centrifugation (4 °C, 4000 rpm, 30 min) and stored over-night at -20 °C. Cells were lysed in 20 mL 1x KlenTaq Lysis Buffer supplemented with 1 mM benzamidine at 37 °C for 15 min, followed by heat denaturation of host proteins at 75 °C for 45 min. Ultra-centrifugation (Ultracentrifuge, L-60) at 40 000 rpm for 60 min at 4 °C was used to remove bacterial cell debris.

Supernatants were incubated with 50 % Ni-IDA sepharose slurry in the presence of 5 mM imidazole for 1.5 h at 4 °C gently shaking. After washing with 30 mL 1x KlenTaq Washing Buffer containing 20 mM imidazol, elution was carried out using 6 mL 1x KlenTaq Elution Buffer. The imidazole was removed using VivaSpin columns and 1x KlenTaq Elution Buffer without imidazole. Size exclusion chromatography was carried out using a superdex 75 column and an ÄKTApurifier. For equilibration, 2 CV of 1x Taq Gelfiltration Buffer were used.

The protein was eluted at 4 °C with a flow rate of 0.5 mL/min in 1 CV (peak between 48-68 mL). Protein was sampled in 1 mL fractions. Fractions were checked by SDS-PAGE and pooled.

The 1x Taq Gelfiltration Buffer was exchanged using VivaSpin columns and 1x KlenTaq elution buffer without imidazole. For storage, glycerol was added to a final concentration of 50 %, (NH4)2SO4 to a final concentration of 16 mM, Tween20 to a final concentration of 0.1 %, Tris·HCl pH 9.2 to a final concentration of 50 mM and MgCl2 to a final concentration of 2.5 mM (1x KlenTaq Storage Buffer). Purified enzymes were stored at -20 °C. Concentration was determined by Bradford Assay or SDS-PAGE.

2.4.2 SDS-PAGE

For SDS-PAGE analysis of proteins, a 4 % stacking and a 12 % separating gel were used.

Protein samples were mixed with 6x SDS-PAGE Loading Dye and heat-denatured at 95 °C for 5 min. Afterwards, 5-20 µL of the protein sample were loaded on the gel and the proteins separated by applying 25-30 mA in 1x SDS-PAGE Running Buffer. Proteins were stained for 30 min using methanol staining solution and destained with coomassie methanol destaining solution for at least 30 min. Alternatively, proteins were stained with coomassie colloidal over-night and destained with coomassie colloidal destaining solution for at least 30 min. As a marker, Unstained Protein Ladder (Fermentas) was used.

2.4.3 Protein Concentration Determination

The Bradford-Assay was employed to determine the protein concentration. Roti‐Quant was diluted with water in a 1:5 ratio and immediately filtrated. 980 µL suspension was then added either to 20 μL of the respective KlenTaq DNA polymerase stored in 1x KlenTaq Storage Buffer or to 20 μL of 0, 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 mg/mL BSA, respectively. The BSA dilution series was used as standard and was prepared in 1x KlenTaq Storage Buffer. After incubation for 5 min, the absorption was measured at 595 nm using the Cary 100 Bio photometer. Protein concentrations were determined by use of the BSA standard curve and verified via SDS-PAGE analysis.