2.2 Methods
2.2.5 Protein Expression
High-cell Density Method and IPTG-Induced Expression.
This expression method, instead of the traditional IPTG-induction expression in M9, was used to reduce the cost of labelled media. E. coli cells were grown in rich medium at 37¶C. Once the cell density reached OD600 ¥ 2, the cell culture was switched to labelled M9 media by gently spinning cells down at 1,500◊g for 5 min. The pellet was repeatedly washed with M9 to remove rich medium. Cells were re- suspended into the concentrated volume of minimal medium (2◊). The cells were cultured for another 1-1.5 h without adding IPTG at 37¶C (OD600 should increase by 1-2 units). IPTG (0.5 mM) was then added to induce protein expression. Cells were then incubated at 15¶C for 20 hours, which was the determined optimal expression time.
Double Colony Selection.
A double colony selection was performed for bacterial expression of triple-labelled protein in D2O (Murray et al. 2012). For the first colony selection, LB- agar plates
incubator at 30¶C. The next day, 9 colonies (with the first colony serving as the negative control) were chosen for expression and a master plate was prepared by streaking the 9 colonies onto it. 8 colonies were induced with 0.5 mM IPTG and expressed at 15¶C in LB with 50% D2O and checked for CPG2 level expression after 24 hours via SDS-PAGE and western blot analysis. The colony with the thickest expression band for CPG2 was selected from the master plate and used to prepare 5 mL overnight culture in LB with 100% D2O at 30¶C. This culture was
spread onto an LB-agar plate with 50% D2O and incubated at 37¶C overnight.
A second round of colony selection was repeated, but this time in LB with 100% D2O. At the end of the procedure, the colony expressing the thickest protein-band
in LB with 100% D2O was selected and used to prepare the final glycerol stock
(800 µL of overnight cell culture of the selected colony from the second master
plate with 400 µL of 50% glycerol), which was stored at -80¶C.
Cell Lysis by Ultrasound Sonication.
Ultrasound sonication was used to disrupt the bacterial cell extracts and lyse cells (5 mL and above) by high frequency sound. Sonicator probes were pre-chilled on ice before use. All samples remained on ice throughout the sonication process. All crude samples (1 mL) and lysed bacterial pellets (10 mL) were subjected to sonication using 6 cycles of 30 seconds on ice followed by 10 seconds of sonication using a 5 mm probe, and 4 cycles of 1 minute on ice followed by 1 minute of sonication using the a 7 mm probe, respectively.
Cell Lysis by Cell disruption.
A cell disruptor was later used to disrupt cell extracts (10 mL and above) at 30 kPSI. In this method, the pressure drops by transferring the cell extract from a chamber at high pressure through an orifice into a chamber at low pressure, ensuring cell lysis. It is fast and efficient but causes significant heating of the sample. For this reason, the collector was always kept at 4¶C before usage.
2.2.6 Protein Purification.
CPG2 Purification.
Recombinant CPG2 was isolated from cell debris by ultrasound sonication or cell disruption. Cell pellets from 1 L culture were thawed on ice and resuspended in 10 mL lysis buffer (20 mM Tris, 137 mM NaCl, no EDTA chelating agent, lysozyme 1 ug/mL, 1 mM AEBSF, pH 8) and incubated at 4¶C for 30 min. The cells were lysed and insoluble fraction pelleted by centrifugation (48,000◊g, 20 mins, 4¶C). The soluble fraction was incubated with 10 mM imidazole and Ni- NTA resin (Novagen, 50% slurry, pre-equilibrated with 10 mM imidazole) for 1 hour. The incubated supernatant-Ni mixture was loaded onto a column and the flow-through collected as the resin set by gravity. The column was washed extensively with 10◊bed volumes of Tris buffer (100 mM NaCl, 20 mM Tris, 10 mM imidazole, pH 8). The his-tagged protein was then eluted with 5◊bed volumes of elution buffer (100 mM NaCl, 20 mM Tris, 250 mM imidazole, pH 8). The buffer was exchanged against Tris buffer (100 mM NaCl, 20 mM Tris, pH 8)
using PD-10 columns to desalt samples.
His-TEV Protease Expression and Purification.
The 2◊YT starter culture (10 mL) was inoculated with cells from glycerol stock and left at 30¶C overnight. This outgrowth was then transferred to a final volume of 1 L fresh 2◊YT to obtain an initial OD600 of 0.1. The cells were then grown at
37¶C until the OD600 reached ¥2, then it was induced with 0.5 mM IPTG and incubated at 15¶C overnight. Recombinant His-TEV protein was then harvested. Cell pellets from 1 L culture were thawed on ice and resuspended in 10 mL Tris lysis buffer (20 mM Tris, 137 mM NaCl, no EDTA — chelating agent, lysozyme 1 µg/mL, 1 mM AEBSF, pH 8) and incubated at 4¶C for 30 min. The cells
were lysed and insoluble fraction pelleted by centrifugation (48,000◊g, 20 min, 4¶C). The soluble fraction was incubated with 10 mM imidazole and Ni-NTA resin (Novagen, 50% slurry, pre-equilibrated with 10 mM imidazole) for 1 hour. The incubated supernatant-Ni mixture was loaded onto a column and the flow- through collected as the resin set by gravity. Column was washed extensively with 10◊bed volume of Tris buffer (100 mM NaCl, 20 mM Tris, 10 mM imidazole, pH 8). The his-tagged protein was then eluted with 5◊bed volume of elution buffer (Tris buffer, 250 mM imidazole, pH 8) and buffer exchanged against Tris buffer (100 mM NaCl, 20 mM Tris, pH 8) using PD-10 columns to desalt samples.
His-Tag Removal by Enzymatic Cleavage.
Both desalted and purified his-tagged CPG2 and TEV proteins in Tris buffer (100 mM NaCl, 20 mM Tris, pH 8) were incubated together for 48 hours at 4¶C for cleavage of the polyhistidine tag.
Purification of the Untagged CPG2.
The overnight CPG2-TEV reaction mixture was incubated with 10 mM imidazole and Ni-NTA resin (Novagen, 50% slurry, pre-equilibrated with 10 mM imidazole) for 1 hour. The incubated supernatant-Ni mixture was loaded onto a column and the flow-through, containing the protein of interest was collected, as the resin set by gravity. The solution volume was slowly reduced using centrifugal units of 20 molecular weight cut-off (Millipore, UK). The unlabelled protein sample was then lyophilised using the freeze drier and stored at -20¶C for ligand-based NMR studies, while the labelled protein sample was used straightaway away for protein-based NMR studies.
2.2.7 Quantification of Protein Concentration.
Protein Concentration Determination using UV-Visible
Spectrophotometer.
60 µL of purified CPG2 was transferred to a Thermo Electron Cuvette and the
using the UV visible BioMate instrument. The concentration was determined using Beer-Lambert Law Equation (2.1), where A is the absorbance, ‘ the molar absorptivity in M-1 cm-1, l the path length in cm and C the concentration in
molar.
A=‘lC (2.1)
Protein Quantification using BCA Assay.
The bicinchoninic acid (BCA) assay was used for colorimetric detection and quantitation of total protein (Thermo Scientific Pierce, UK). The manufacturer’s guidelines were followed to perform the assay. After incubation at room temperature (i.e. 25¶C) for 2 hours, the absorbance reading of the sample of interest and the standards at 562 nm were taken using the UV- visible spectrophotometer. Set at 562 nm, the instrument was zeroed using a cuvette filled with water. The average absorbance reading for standard I (i.e. blank) was subtracted from the absorbance reading for each remaining standard and unknown sample. The standard curve was prepared by plotting the average blank-corrected absorbance reading for each standard versus their respective concentrations in µg/mL. Using the standard curve, the gradient was be
calculated and, using Equation (2.2), the protein concentration was established.