2.2 Methods
2.2.8 Protein Detection
Protein Solubility Assay.
Sonicated crude samples (1 mL) were centrifuged (5,000◊g) for 5 min to pellet the insoluble components. The supernatant (soluble fraction) and the pellet (the insoluble fraction) were retained for SDS-PAGE analysis. 10 mL sonicated lysed samples were also centrifuged (5,000◊g) for 15 min at 4¶C. The supernatant and the pellet were retained for further purification.
Optical Density of Culture Samples.
Sample was diluted to yield an OD600 reading between 0.1 and 0.8. Usually a
1:10 dilution of the culture was sufficient. The same media used for growth of the culture was used for zeroing the spectrophotometer and diluting the samples for taking OD600 readings. The dilution factor and the OD600 reading were recorded
to calculate the actual OD600 of the culture.
Normalising SDS-PAGE Samples.
To accurately compare the amount of protein present between various culture samples taken at various growth stages, the samples were normalised according to the cell density (OD600) at the time the samples were taken. Maximum volumes
that can be loaded into the wells of the SDS-PAGE gel were also taken into account. The volume of samples loaded onto the gels was normalised with respect to the concentration of cells in the pre-induction sample.
SDS-PAGE Gels.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for the separation of proteins based on their molecular weight. All SDS- PAGE gels were prepared using the standard Laemmli Method (Laemmli 1970).
Table 2.8: SDS-PAGE gels sample buffer and running buffer. (a) Sample Buffer SDS 10% w/v Dithiothreitol 10 mM Glycerol 20% v/v Tris-HCl, pH 6.8 0.2 M Bromophenolblue 0.05% w/v (b)Running Buffer Tris-HCl 25 mM Glycine 200 mM SDS 0.1% w/v
The percentage acrylamide was selected based on the molecular weight range of proteins we wished to separate (Table 2.8). A 12% gel was suitable to separate protein in the 10 kDa-200 kDa range (see Table 2.9). Tetramethylethylenediamine (TEMED), an essential catalyst for polyacrylamide gel polymerisation, and ammonium persulfate (APS), an oxidising agent that is used with TEMED to catalyse the polymerisation of acrylamide, were not added until the gel was ready to be poured into the gel casting plates.
Table 2.9: SDS-PAGE gel recipe. (a)Resolving Gel Solution (12% acrylamide)
12% H20 10.2 mL 1.5 mM Tris-HCl, pH 8.8 7.5 mL 20% SDS (w/v) 0.15 mL 30% Acrylamide / 0.8% bis-acrylamide (w/v) 12 mL 10% (w/v) ammonium persulfate 0.15 mL 0.15 mL TEMED 0.02 mL
(b)Stacking Gel Solution (4% acrylamide) 4% H20 3.075 mL 0.5 mM Tris-HCl, pH 6.8 1.25 mL 20% SDS (w/v) 0.025 mL 30% Acrylamide / 0.8% bis-acrylamide (w/v) 0.67 mL 10% (w/v) ammonium persulfate 0.025 mL TEMED 0.005 mL SDS-PAGE Analysis.
The Mini Protean 3 Electrophoresis System (Bio-Rad) was used to perform all electrophoresis according to the manufacturer’s instructions.
1 mL crude samples and insoluble fractions from sonicated samples were centrifuged (5,000◊g) for 5 min at 4¶C and the supernatant were discarded. Pellets were resuspended in 15 µL of PBS buffer and 5 µL Laemmli Buffer.
Sonicated soluble samples (1 mL, 10 mL) were prepared for SDS by adding 5µL
heated at 90¶C on a heating block for 5 min exactly. The sample was centrifuged (5,000 rpm) and vortexed prior and after heating for 5 min. The volume sample of SDS were normalised with respect to the concentration of cells in the pre-induction sample, and ready for SDS-PAGE analysis. SeeBlue® Plus2 Pre-Stained Standard (Invitrogen, 4-250 kDa) or ColorPlus restained Protein marker Broad range (NEB, 7-175 kDa) were run on gels as protein standards. Gels were run at 125 V for approximately 15 min and further at 200 V for 35 min until the front dye had migrated to the end of the gel. Upon completion, the gel was subjected to Coomassie Blue Staining overnight on a rocker. Gel was then destained with destaining solution (7% acetic acid, 40% ethanol) until the bands were clearly visible and scanned (gels were maintained between 2 plastic sheets) and stored in H2O.
Coomassie Staining.
Upon completion, the gel was subjected to coomassie blue staining overnight on a rocker. Gels were first immersed in 20 mL of fixing solution [50% (v/v) MeOH, 10% (v/v) acetic acid] for 15 min, then stained in 20 mL coomassie solution [0.025% (w/v) coomassie blue R250, 10% (v/v) MeOH] for 4 to 12 hours. Gels were destained in a 10% (v/v) acetic acid solution until the bands were clearly visible.
Western Blot.
Gels were electro-blotted onto a 0.45 mm pore size nitrocellulose membrane at 100 V, 35 mA for 60 min using the Mini Trans-Blot® Electrophoretic Transfer Cell according to the manufacturer’s guide (Bio-Rad).
After the transfer, the membrane was blocked in Tris-Buffered Saline and Tween-20 (TBST) (30 mM Tris-HCl, 140 mM NaCl, 3 mM KCl, 0.1 % Tween- 20, pH 7.4) and 5% milk solution for maximum 1 hour at room temperature (or overnight at 4¶C), then washed with TBST buffer 3 times for 5 min. The membrane was inoculated for 1 hour with the primary antibody mouse anti- polyHistidine, which was diluted in TBST and 5% milk, and then the washing steps were repeated (3◊5 mins in TBST). The secondary antibody tablet, anti- mouse anti-polyHistidine-alkaline phosphatase (Sigma), diluted in 10 mL distilled H2O and used to incubate the blot for 1 hour, followed by 2◊30 min washing steps
with TBST.
Size exclusion size exclusion chromatography analysis.
4 mg/mL of protein solutions (CPG2, CPG2CAT, CPG2CAT’and CPG2DIM) were
prepared in buffer A (20 mM Tris, 100 mM NaCl, pH 8). Gel filtration chromatography was performed on a HiLoad 16/600 Superdex 75 g GL column by fast protein liquid chromatography ÄKTAFPLC (GE Healthcare UK Ltd. Buckinghamshire, UK) at 4°C and at a flow rate of 0.5 mL/min. The column was equilibrated with two column volumes of buffer A. The detection was performed by monitoring the column eluate at 280 nm. To determine the
molecular weight, a parallel column was run with the following molecular weight markers at 5 mg/mL concentration each: —-amylase (200 kDa), Albumin (66 kDa) and Cytochrome C (12.4 kDa) (see Figure 2.1).
Figure 2.1: Elution and Calibration curve obtained with —-amylase, Albumin and Cytochrome C protein Standards run on HiLoad 16/600 Superdex 75 g GL column.
A)The graph shows elution profile from HiLoad 16/600 Superdex 75 g GL column for calibration standards (—-amylase 200 kDa, Albumen 66 kDa, Cytochrome C 12.4 kDa) as absorption in
milli-absorption units (mAU) at 280 nm versus volume in milliliters (mL). An isocratic gradient of 100% of buffer A (20 mM Tris, 100 mM NaCl, pH 8) was used. B)The calibration curve graph represents the gel phase distribution coefficient Kav versus the molecular weight (MW).
Kav= (Ve-Vo)/(Vc-Vo), where Ve= elution volume, Vo= column void volume, Vc= geometric
column volume. Straight line is the calibration curve calculated from the data for molecular weight standards. Using the equation for the line, values for the molecular weights of CPG2, CPG2CAT, CPG2CAT‘ and CPG2DIM were calculated (see Chapters 2 and 6).
Protein identification by Means of Tryptic Digest and Nano LC-ESI- MS/MS.
Excised bands from coomassie stained gel pieces were processed and tryptically digested using the manufacturer’s recommended protocol on the MassPrep robotic protein handling system Facility1. The extracted peptides from each
sample were analysed by means of nano LC-ESI-MS/MS using the NanoAcquity/Ultima Global instrumentation (Waters) using a 30 minutes LC gradient. All MS and MS/MS data were corrected for mass drift using reference data collected from the (Glu1)-Fibrinopeptide B (human-F3261 Sigma) sampled each minute of data collection. A full description of the methodologies employed is available on request. The data were used to interrogate the E. coli BL21 database (http://www.ebi.ac.uk/integr8) appended with the sequence supplied, using ProteinLynx Global Server v2.5.1.
Identification of proteins via MaXis II
All his-tagged proteins were analysed for their identity under positive-mode using liquid chromatography electrospray ionisation ultra-high resolution quadrupole time-of-flight mass spectrometry (LC-ESI UHR QTOF)2. Samples
at 20 µM protein in 20 µM ammonium bicarbonate where diluted by 1 in 5 in
30% acetonitrile (ACN) with 0.1% formic acid (FA), and 40 µM aliquots of
these were loaded onto a C4 column with 100% water and 0.1% FA as solvent A
1Dr Suzanne Slade (School of Life Sciences, University of Warwick) performed tryptic digest,
protein identification and analysis by Nano LC-ESI-MS/MS.
2Philip Aston (Department of Chemistry, University of Warwick) performed analysis by
and 100% ACN with 0.1% FA as solvent B using a 3-step gradient (75% of solvent A for 16 min, 100% of solvent B for 5 min and 75% of solvent A for 11.5 min) and eluted directly into the Bruker MaXis II mass spectrometer. The resulting total ion count (TIC) data (summed intensity across the entire range of masses being detected at every point in the analysis) was extracted and mass averaged in the region containing the proteins of interest, and deconvoluted using the Data Anaysis Software (Bruker Daltonik GmbH; Bremen, Germany).
To confirm the identity of expressed proteins, samples were analysed before TEV digestion (i.e. with the presence of his-tag), by mass spectrometry. Resulting spectra were averaged (see Table 2.10). Full-length his-tagged CPG223-415, CPG2CAT and CPG2DIM were identified at the exact molecular
mass predicted ± 1 Dalton, containing the gene sequence. For CPG2 the [M+H]+ ion was observed at 45477.9 Da (expected molecular ion mass including
the his-tag is 45478.6 Da), for CPG2CAT at 33889.7 Da (expected molecular ion
mass is 33889.4 Da) and for CPG2DIM at 15649.1 Da (expected molecular ion
mass is 15649.7 Da). For his-tagged CPG2CAT’, the [M+H]+ ion was observed
at 22922.9 Da (expected molecular ion mass is 22580.6 Da) with 342.3 Da of excess mass, which appeared to show post-translational modifications of phosphogluconoylation of His-tag in E.coli (258 Da), and a combination of multiple post-translational modifications (i.e. acetylation + 42 Da) at different sites (i.e. different amino acid such as Ser, Thr or Lys) could also explain the remaining 84 Da of excess mass (Cain et al. 2014). The spectra showed some impurities remaining after the first affinity chromatography, but the highest intensity peak was the [M+H]+ peak. All CPG2
identified at the exact molecular mass predicted ± 1 Dalton, except for E175A and E200A, which were at the molecular mass predicted ± 2 Daltons.
Table 2.10: Mass average of all expressed constructs (without TEV protease cleavage) obtained from LC-ESI UHR QTOF mass spectrometry.
Constructs Calculated Mass
(Da)
Observed Mass (Da)
Mass Shift (Da)
CPG223-415 45478.6 45477.9 -0.7 CPG2CAT 33889.4 33889.7 +0.3 CPG2CAT’ 22580.6 22922.9 +342.3 CPG2DIM 15649.7 15649.1 -0.6 H112A mutant 45412.6 45411.7 -0.9 H385A mutant 45412.6 45411.8 -0.8 E175A mutant 45420.6 45418.7 -1.9 E200A mutant 45420.6 45418.7 -1.9 D141A mutant 45434.6 45433.7 -0.9 K208A mutant 45421.5 45420.8 -0.7 S210A mutant 45462.6 45461.8 -0.8 T357A mutant 45448.6 45447.7 -0.9 Mass Spectrometry.
A simple protocol for the analysis of intact proteins by matrix-assisted laser-desorption / ionisation time-of-flight mass spectrometry (MALDI-TOF MS), utilising a mixture of sinapinic matrix. The degree of isotope incorporation after labelling (i.e. e.g. 13N/13C/2H labelling), his-tag removal by
TEV protease and sample homogeneity were also rapidly analysed using MALDI-TOF MS on an Autoflex Speed instrument (Bruker, UK). The protein samples were buffer exchanged into 20 mM ammonium acetate using Amicon
Ultra centrifugal filters with a 20 kDa MWCO (Millipore, UK). One microliter of the protein solution was spotted onto the target MALDI plate and then overlaid with two microliters of the matrix consisting of a saturated solution of sinapinic acid or 2-Cyano-3-(4-hydroxyphenyl) acrylic acid (Sigma, UK) in 50% acetonitrile (Sigma, UK) and 2.5% trifluoroacetic acid (Sigma, UK). The data was acquired with FLeX Control software and processed using FLeX Analysis software by Bruker (Coventry, UK).