4.2.1 Preparation of cell pellets for protein purification
5 g pellets of frozen E. coli cells were diluted with 50 ml of buffer A (running buffer for HisTrap) and re-suspended by vortexing. Small amounts of DNase and lysozyme were added. The pellets were incubated at 4 °C shaking for 30 min. Then, the resuspension was applied to sonication for 15 x 1 min, with 1 min of break between the cycles. During sonication, the cell resuspension was incubated on ice.
Subsequently, the sample was centrifuged at 15000 g for 20 min to pellet cell debris. Then, the supernatant was collected and used for loading of a respective affinity‐chromatography column.
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4.2.2 Protein purification
For purification of heterologously produced protein from E. coli, the principle of nickel affinity chromatography (NAC) was used in this study. The coding sequence of the protein was cloned into E.
coli expression vector (pET-28b), resulting in the production of the protein fused to 6xHis‐ tags. The 6xHis‐tagged protein was immobilized at Ni2+‐NTA‐agarose resin. The protein purification steps were either conducted on an ÄKTAprime™ plus or an ÄKTAFPLC™ system (GE Healthcare, USA). The protein sample was loaded onto the NAC column with a flow rate of 1.5 ml min-1, and washed with 5% of buffer B (elution buffer) in a volume of 20 ml. Then, the column was washed with 40% of buffer B, and finally with 100% of buffer B. The compositions of buffers used are shown below.
Table 4.22 Buffer A for HisTrap Table 4.23 Buffer B for HisTrap
Component Concentration Component Concentration
Tris 0.02 M Tris 0.02 M
NaCl 0.15 M NaCl 0.15 M
PMSF 1 ml Imidazole 0.5 M
pH=8 pH=8
4.2.3 Size exclusion chromatography (SEC)
Further purification of affinity chromatography‐derived proteins and determination of the enzymes’
multimeric states were achieved by size exclusion chromatography (SEC). The SEC column was firstly washed with one column volume of H2O and then with one column volume of SEC buffer. The protein sample was loaded onto the SEC column with a flow rate of 1 ml min-1. Then, fractions were collected in a size of 5 ml, starting at an elution‐volume of 90 ml after sample injection. Molecular weight of eluted protein was determined according to the equation shown in Figure 4.1. In this study, for protein purification, two kinds of buffer were used as the SEC buffer. Their compositions are shown below.
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Figure 4.1 Standard curve for determination of molecular weights according to SEC. Standard curve was determined by Dr. Florian Brodhun (Department of Plant Biochemistry, University of Göttingen, Germany) according to elution volumes of HiLoad 26/60 Superdex S200 SEC‐column.
Table 4.24 Tris-HCl buffer for SEC Component Concentration
Tris 0.05 M
NaCl 0.15 M
Glycerol 2%
pH=7
Table 4.25 Phosphate buffer for SEC
Concentration Component
0.05 M Phosphate buffer = 39 ml 1 M NaH2PO4 + 61 ml 1 M Na2HPO4
0.15 M NaCl
30 mg 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS)
pH=7
4.2.4 Desalting chromatography
The protein purified from affinity chromatography was load onto HisTrap Desalting Column (GE Healthcare, USA) to remove imidazole according to the manual provided by the manufacturer, protein was eluted in TBS buffer for further assay.
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4.2.5 SDS polyacrylamide gel electrophoresis (SDS‐PAGE)
SDS‐PAGE was performed according to the method described by Weber (1977). For SDS‐PAGE, protein samples were mixed with 4 x sample loading buffer and water, denatured at 95 °C for 5 min. 5 μl of denatured protein samples and 5 μl of a MW marker were loaded onto the SDS gel. SDS‐PAGE was run on Mini‐PROTEAN3 electrophoresis Systems at 35 mA and 300 V until the bromophenol‐blue front migrated out of the gel. Proteins were stained in Coomassie‐staining solution at room temperature for 2 h while shaking, then the gel was unstained with Destaining Buffer. Additionally, the SDS gel was supplemented with 2,2,2‐trichloroethanol (TCE) according to Ladner et al. (2004) in order to achieve first, fast visualization of the separated proteins on the gel. Respective gel was developed for 2 min under UV‐radiation at 312 nm. Afterwards, the gel was exposed to UV‐light for 20 s while the image was recorded upon utilization of the SybrGreen‐filter in a Diana machine.
Table 4.26 Stacking gels (4%) for SDS-PAGE
Component Amount
ddH20 6.12 ml
0.5 M Tris/HCl, pH = 6.8 2.5 ml
Acrylamide bis-acrylamid (30% of acrylamid, w/v) 1.3 ml
APS (25%, w/v) 400 μl
TEMED 100 μl
Table 4.27 Separation gels (12%) for SDS-PAGE
Component Amount
ddH20 5.52 ml
Acrylamide bis-acrylamid (30% of acrylamid, w/v) 6.4 ml
0.5 M Tris/HCl, pH = 8.8 4.00 ml
APS (25%, w/v) 64 μl
TEMED 16 μl
59 Table 4.28 The composition of 10 x gel running buffer
Concentration Component
200 mM Glycine
25 mM Tris/HCl, pH = 8.0
0.1% (w/v) SDS
Table 4.29 The composition of 4 X sample loading buffer
Component Amount
1 M Tris HCl pH 6.8 1.5 ml 1 M DTT (dithiothreitol) 3 ml
SDS 0.6 g
Bromophenol blue 0.03 g
Glycerol 2.4 ml
Table 4.30 The composition of staining buffer
Table 4.31 The composition of destaining buffer
Component Concentration Component Concentration
H20 50% (v/v) H2O 60% (v/v)
Ethanol 40% (v/v) Ethanol 30% (v/v)
Acetic acid 10% (v/v) Acetic acid 10% (v/v)
Coomassie Brilliant Blue G250
0.5% (w/v)
4.2.6 Measurement of protein concentration
The concentrations of protein were determined using the method described by Bradford (1976). A respective calibration curve was prepared with BSA as the standard in a linear range of 20 μg ml-1 to 1000 μg ml-1. For preparation of the calibration curve as well as for determination protein
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concentration of unknown samples, 1 ml of Bradford‐Reagent was mixed with 20 μl protein sample and briefly vortexed. After incubation in the dark for 10 min, the absorption of sample was measured at 595 nm in a spectrophotometer. 1 ml of Bradford‐Reagent without any sample was used as a blank‐
value.
Table 4.32 The composition of bradford‐reagent
Component Amount
Serva Blue G 35 g
Ethanol 25 ml
85% Phosphoric acid 50 ml
H20 425 ml
4.2.7 DTNB‐based in vitro test of acyltransferase activity
The activity of WS was tested according to the method described by Willis et al. (2008). DTNB‐based in vitro assay was used to monitor the enzyme‐mediated cleavage of acyl‐CoA‐derived thioester‐bonds.
The enzyme catalyzes the cleavage of acyl‐CoA resulting in the liberation of free CoA‐SH molecule.
Free CoA‐SH reacts with the intramolecular disulfide bond of a DTNB molecule, therefore the disulfide bond is cleaved. As a result, a disulfide consisting of a CoA and a 5‐thio‐2‐nitrobenzoic acid (TNB) is generated. At the same moment, a free TNB2‐ion is released, which absorbs at 412 nm and appears yellow.
Figure 4.2 Reaction scheme of the DTNB‐reaction. DTNB is cleaved, forming a mixed disulfide with free CoA molecules and a TNB2‐ ion which absorbs at 412 nm.
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The reaction sample contained 0.2 mg ml-1 DTNB, the same amount of fatty alcohol and fatty acyl‐CoA in a total volume of 1 ml TBS. The sample was pipetted in polystyrene cuvettes without enzymes and vortexed for 5 s. Cuvettes were placed in a Cary 100 Bio spectrophotometer (Varian, Germany) and the measured absorbance was taken as a blank‐value. After absorbance stabilized to a continuous baseline, the enzyme was added, then the reaction sample was vigorously mixed and the increased absorbance was continuously recorded at 412 nm.