2. Materials and Methods
2.2. Methods
2.2.7. Preparation and detection of proteins
Overexpression of recombinant proteins in E. coli
The E. coli BL21 strain with the relevant plasmid was used to inoculate an overnight culture. The main culture of 1 l LB was inoculated to an OD600 of 0.1 and grown at 37°C and 200 rpm until an optical density of 0.6 to 0.8 was reached. At this point, the inducer isopropyl-β-D-thio-galactopyranoside (IPTG) was added in a final concentration of 1 mM.
gene of interest kanR
Chromosome with cre recombinase
+ Xylose
A
B
kanR
Lox72
30
Cell disruption with French pressure cell press
A cell pellet was resuspended in buffer W or ZAP buffer and filled into the precooled bomb. The remaining air was removed before the bomb was locked and placed in the French press. The disruption was performed three times with a pressure of 18.000 PSI.
Preparation of crude extracts for β-galactosidase activity assay)
The cell pellet, from a cell culture grown to an OD600 of 0.5-0.8, was resuspended in 400 µl Z-buffer with β-mercaptoethanol and Lysozyme/DNase I mix (60 µl on 12 ml Buffer). The samples were incubated 10 min at 37°C and 600 rpm and afterwards centrifuged 3 min at 4°C and 14800 rpm.
LD mix 100 mg Lysozyme
10 mg DNase I Ad to 10 ml with dH2O
Z-Buffer 0.534 g Na2HPO4 x 2 H2O
0.276 g NaH2PO4
0.037 g KCl
50 µl 1 M MgSO4
175 µl β-mercaptoethanol Ad to 50 ml with dH2O
Purification of Strep-tagged proteins
A cell pellet of the respective E. coli strain was resuspended in cold buffer W and the cells were disrupted with the french pressure cell press as described above. The solution was centrifuged at 35000 rpm and 4°C for 30 min to remove cell debris. This crude extract was then loaded onto pre-equilibrated 500 µl Strep-Tactin Sepharose (IBA) in a Poly-Prep chromatography column (Biorad).
Buffer W was used to wash the mixture five times and the bound proteins were then eluted with buffer E in four fractions. The fractions were analysed by SDS page (Blötz et al., 2017).
Buffer W (pH 8.0) 121.14 g Tris-base 87.7 g NaCl 3.72 g Na2EDTA Adjust the pH with HCl to 8.0 Ad to 1000 ml with dH2O
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Buffer E 0.027 g D-Desthiobiotin
Add 50 ml 1x buffer W
Purification of His-tagged proteins
An E. coli cell pellet was resuspended in ZAP Buffer and the cells were disrupted as described above.
To remove cell debris, the cell solution was centrifuged at 17.500 rpm and 4°C for 30 min. 1.25 ml of Ni-NTA® sepharose was loaded onto a column and equilibrated with 12.5 ml ZAP Buffer. The crude extract was loaded onto the column and the flow through was collected. Five washing steps were performed with each 10 ml ZAP buffer and the elution was done with ZAP buffer with increasing concentrations of imidazole (Blötz et al., 2017).
10x ZAP Buffer 60.57 g Tris-base 116.88 g NaCL
Adjust the pH with HCl to 7.5 Ad to 1000 ml with dH2O
Dialysis
The dialysis was used to remove the desthiobiotin from the protein solution or to change to a desired buffer. Therefore, the elution fractions with the highest protein amount were pipetted into a dialysis tube and dialyzed against the desired buffer in an excess of 1000 fold overnight.
Denaturing gel electrophoresis of proteins (SDS-PAGE)
The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method described by Laemmli (1970) was used to analyse the protein sizes. First the protein samples were mixed with 5x SDS loading dye and denatured for 10 min at 95°C. The SDS gels consist of a stacking gel with 5%
polyacrylamide and a running gel with 12% polyacrylamide underneath. The samples and a protein size marker PageRuler™ Plus prestained were loaded onto the gel and the electrophoresis was performed at 100-160 (Blötz et al., 2017).
5x SDS loading dye 1.4 ml Tris-HCl (1 M, pH 7.0) 3 ml Glycerol (100%)
2 ml SDS (20%)
1.6 ml β-Mercaptoethanol (100%) 0.01 g Bromphenol blue
2 ml dH2O
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10x Page buffer (PLP) 144 g L-glycine
30 g Tris-base
10 g SDS
12% running gel 4.8 ml dH2O
3.9 ml Tris-HCl (1.5 M, pH 8.8) 6 ml Acryl-bisacrylamide (30%) 150 µl SDS (10%)
150 µl Ammonium persulfate (10%)
15 µl TEMED
5% stacking gel 10.25 ml dH2O
1.305 ml Tris-HCl (1.5 M, pH 6.8) 1.95 ml Acryl-bisacrylamide (30%) 150 µl SDS (10%)
150 µl Ammonium persulfate (10%)
30 µl TEMED
Coomassie staining
After the SDS-Page, the protein gels were stained with Coomassie Brilliant Blue. Therefore, the gels were first treated with a fixation solution for 30 min at RT. A staining solution was used to stain the proteins. Afterwards, the gels were destained with water (Blötz et al., 2017).
Fixation solution 10% Acetic acid
50% Methanol
Ad to the final volume with dH2O
Staining solution 0.5% Coomassie brilliant blue
10% Acetic acid
45% Methanol
33 Enzyme activity assays
β-galactosidase activity assay
For the measurement of the β-galactosidase activity, 100 µl of the crude extract was mixed with 700 µl Z-Buffer with β-mercaptoethanol and incubated for 5 min at 28°C. The reaction was initiated with the addition of 200 µl o-nitrophenyl-β-D-galactopyranoside (ONPG). The reaction is stopped with 500 ml 1M Na2CO3 when the solution turns yellow. To detect the amount of produced o-nitrophenyl, the OD420 of the samples was measured. 10 µl of the crude extract was used for the Bradford assay to determine the protein concentration. The β-galactosidase activity was calculated with the following formula:
𝑂𝐷 420𝑛𝑚
𝛥𝑡 × 𝑂𝐷 595𝑛𝑚× 2005.3475
ONPG 4 mg o-Nitrophenyl-β-D-Galactopyranoside
1 ml Z-Buffer without β-mercaptoethanol
1 M Na2CO3 26.5 g Na2CO3
Ad to 250 ml with dH2O
Citrate synthase activity assay
For the determination of citrate synthase activity, a colorimetric assay was performed. The citrate synthase converts acetyl-CoA and oxaloacetate to citrate and coenzyme A. This coenzyme A reacts in the assay with the Ellman’s reagent 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) and the resulting compound TNB can be measured spectrophotometrically at 412 nm. The reaction mixture contains 100 mM Tris-HCl (pH 8.0), 1 mM DTNB and 50 nM of the purified citrate synthases CitZ or CitA. The reaction was initiated by the addition of 0.3 mM oxaloacetate and 0.3 mM acetyl-CoA and the absorbance at 412 nm was measured at 25°C. For the determination of Km and Vmax values, one substrate was added in a constant concentration and the other substrate was added in varying concentrations (0.03-0.45 mM) (Ellman, 1959; Johansson and Pettersson, 1974).
From the resulting data, the initial reaction rate vo was determined as the change of absorption (ΔA) per minute. The next step was to plot the values 1/vo against 1/substrate concentration [S] in a Linewaever-Burk diagram (Lineweaver and Burk, 1934). From this plot, the Km and Vmax values can be determined by the following equation:
1
𝑣0= 𝐾𝑀
𝑉𝑚𝑎𝑥 [𝑆]+ 1 𝑉𝑚𝑎𝑥
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Hom assay
For the measurement of Hom enzyme activity, the reverse reaction from homoserine to L-aspartate 4-semialdehyde was used. The simultaneous conversion of NADP+ to NADPH and the accompanying change in absorption at 340 nm was measured with a photometer. The reaction mixture contains 100 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM NADP+ and 0.02 mM DTT. To initiate the reaction, 10 mM homoserine was added to the reaction. The change in absorption at 340 nm was measured at 25 or 37°C (Hama et al., 1990; Hama et al., 1991).
Preparation of samples for proteome analysis
A 4 ml overnight culture was used to inoculate a 50 ml LB preculture to an OD600 of 0.05 and incubated at 37°C and 200 rpm until an OD600 of 0.5 is reached. From this preculture, a main culture of 150 ml in a 1 l flask was inoculated to an OD600 of 0.05. After incubation to an OD600 of 0.5, 30 ml of the cells were harvested by centrifugation at 4°C for 15 min and 8500 rpm. The supernatant was removed and the cells were washed in 10 ml TE Buffer. The solution was again centrifuged as described and the supernatant was removed. The samples were frozen in liquid nitrogen and analysed by the Department of Functional Genomics in Greifswald. The analysis was performed as described in Reuß et al. (2017).