Feb 10, 2023
Purification of 10xHis-SuperTEV
- Kelvin Lau1,
- Bouchra Bouchri2,
- Florence Pojer2
- 1EPFL - EPF Lausanne;
- 2EPFL SV PTPSP
Protocol Citation: Kelvin Lau, Bouchra Bouchri, Florence Pojer 2023. Purification of 10xHis-SuperTEV . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2zxng3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 08, 2023
Last Modified: February 10, 2023
Protocol Integer ID: 76660
Keywords: TEV, protease, purification, bacteria, TEV protease, protein purification
Funders Acknowledgement:
EPFL
Grant ID: SV Core Funding
Abstract
SuperTEV is a mutated version of TEV (Tobacco Etch Protease) a cysteine protease widely used in labs as it is highly specific to a cleavage sequence that can be genetically encoded. Depending on the lab, it has been reported that the protease is unstable or purifies with low yields. Efforts have been made for many years (Tropea, 2009, Methods Mol Biol) to improve its solubility. Here we report our platform's efforts in generating a new TEV variant, called SuperTEV, that incorporates 9 mutations identified by different groups in recent years applied together at once. We find that the protein is easily produced and purified in large amounts and is functional.
The list of mutations on the canonical TEV protease are the following (with references that also identified the same mutations):
T17S (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012
L56V (rational design, improved solubility) Cabrita et al., 2007 and Wei et al., 2012
N68D (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012
I77V (directed evolution, increased solubility and production) van den Berg et al., 2006 and Wei et al., 2012
S135G (rational design, increased solubility) Cabrita et al., 2007 and Wei et al., 2012
I138T (increased catalytic activity; TEV3) Sanchez and Ting, 2019
S153N (increased catalytic activity; TEV3) Sanchez and Ting, 2019
T180A (increased catalytic activity; TEV3) Sanchez and Ting, 2019
S219V (inhibits autoproteolysis) Kapust, 2001
References:
Wei et al., 2012. Protein Expression and Purification
Sanchez and Ting, 2019. Nature Methods
van den Berg et al., 2006. Journal of Biotechnology
Cabrita et al., 2007. Protein Science
Kapust et al., 2002. Biochemical and Biophysical Research Communications
Guidelines
This protocol is written with the expectation of standard bacterial culture and basic purification knowledge
Materials
Plasmid Addgene #193833 (https://www.addgene.org/193833)
BL21 (DE3) cells (Lucigen)
AutoTB + trace elements (Formedium) or TB + trace elements (Formedium)
Glycerol (Applichem)
AktaGO (Cytiva)
HiFliq Ni-NTA columns (ProteinArk)
12-14 kDa dialysis tubing (SpectraPor)
Dialysis clips (SpectraPor)
Emulsiflex
5 M NaCl
1 M HEPES, pH 7.5
2.5 M imidazole pH 7.5 (We recommend, Sigma #56749-1KG for low background absorbance)
1 M DTT
1 M IPTG
4X LDS loading dye (Genscript)
NuPage 4-20% SDS-PAGE Gels (Thermofisher)
8M Urea
Before start
Recommended to have ready before starting alongside Materials.
-Autoclaved flasks
-Autoclaved LB media
-Autoclaved AutoTB media
-Buffers
-Liquid nitrogen
Growing bacterial cultures (6 L)
Growing bacterial cultures (6 L)
4d
4d
Transform the bacterial plasmid expressing the 10xHis-SuperTEV into BL21 (DE3) cells. Plate on to LB-Agar plates + Kanamycin. Grow overnight at 37 °C or over the weekend at 25 °C .
1d
In an afternoon, pick a streak of cells and innoculate into 200 mL of LB + Kanamycin media. Note: for every 1 L of expression culture, you will require 10-20 mL of pre-culture. Grow overnight at 37 °C
1d
The next morning, innoculate 20-40 mL of preculture into 3 flasks containing 2 L of Autoinduction TB media (2 L in a 5 L flask is appropriate). Shake in an incubator at37 °C for 3-4 hours until OD600 ~0.8-1. Take a 1 mL sample of the culture. Immediately change the temperature to 18 °C and continue shaking overnight (approximately 20:00:00 )
Note
Regular LB or TB media can also be used. Induction will be at the same point as the temperature change with 0.5 mM IPTG
20h
Take a 1 mL sample of the culture and measure the OD600. Harvest the culture by centrifuging at 5000 x g, 10°C, 00:30:00 . Transfer the pellets to 50 mL tubes directly or resuspend in minimal amounts wash buffer buffer transferring. Pellets can be used immediately or store at -20 °C indefinitely.
30m
Confirm expression by running an SDS-PAGE gel to observe the appearance of a band around 25 kDa that would represent the production of the 10xHis-SuperTEV.
Recommended recipe to prepare SDS-PAGE samples. Mix 1:4, 4X LDS loading dye and 8 M Urea, to make a Urea loading dye.
For samples, calculate the amount of sample needed to prepare. We use the following formula:
1/OD600 x 100 uL = volume in uL to centrifuge down 14000 rpm, 00:01:00 .
Discard the supernatant, Keep the pellet.
Resuspend pellet in 40 uL of the Urea loading dye.
Load 10 uL on to a NuPage 4-12% bis-Tris SDS-PAGE gel.
Expected result
Observed appearance of band near 25 kDa that would represent the induced protein
Induction gel
40m
Prepare purification buffers
Prepare purification buffers
30m
30m
From stock solutions prepare the following :
Filter 0.22 or 0.45 um
| A | B | C | |
| Buffer | Component | Concentration (mM) | |
| Wash buffer (2 L) | NaCl | 700 | |
| HEPES, pH 7.5 | 20 | ||
| Elution buffer (1 L) | NaCl | 700 | |
| HEPES, pH 7.5 | 20 | ||
| Imidazole, pH 7.5 | 500 |
Buffer lists for purification
30m
Purification by Ni-NTA on an AKTA system
Purification by Ni-NTA on an AKTA system
3h 30m
3h 30m
Resuspended pellets were supplemented with glycerol to 10% and DNase.
The mixture was then lysed using an Emulsiflex device by 3 passes until the lysate was visually not viscous.
Note
Other lysis methods such as sonication and french press can also be used. We find it most efficient with an emulsiflex as due to the larger culture volumes, pellets are large and there is substantial amounts of nucleic acids that need to be sheared.
30m
Total volume is around100 mL . Centrifuge down the lysate at 20000 x g, 4°C, 00:40:00 .
40m
Transfer the supernatant to a clean container, being sure not to transfer any liquid that does not contain visually turbid particles near the pellet. Filter the supernatant using a 0.45 um filter
15m
Supplement the filtered supernatant with 1 Molarity (M) imidazole to a final concentration of 25 millimolar (mM) imidazole
5m
Note
Bulk resin can also be used. One can program their AKTA system to equilibrate and load all automatically or be run manually as per one's lab methodology.
See the Materials for our recommendation for low-absorbance imidazole that has minimal interference with A280 measurements of protiens
Using an AKTA system, load the sample on to 3 x 5 mL HiFliq Ni-NTA (15 mL = 1 CV) columns equilibrated with 5% Elution buffer (25 millimolar (mM) imidazole ).
The loaded sample was then washed extensively and eluted as a step gradient as follows
| A | B | C | |
| Step | Elution buffer concentration (%B) | Column Volumes (CV) | |
| Wash + 25 mM imidazole | 5 | 8 | |
| Wash + 100 mM imidazole | 20 | 5 | |
| Wash + 500 mM imidazole | 100 | 10 |
Gradient for Elution
Expected result
A typical elution profile looks like this
Expected peak during elution
Collect fractions as appropriate for verification on an SDS-PAGE gel.
Expected result
Typical SDS-PAGE gels of all fractions during purification
SDS-PAGE gel of Ni-NTA purification
1h 30m
The protein elutes at 500 millimolar (mM) imidazole . Fractions containing the purest protein are pooled. It is advised to measure the absorbance at A280 to determine the concentration.
1 mg/mL of SuperTEV = A280, 1.2
10m
Dilute with wash buffer to around 2-3 mg/mL or as desired.
5m
Transfer the protein to dialysis bags for dialysis in 2 L of dialysis buffer ( 150 millimolar (mM) NaCl , 20 millimolar (mM) HEPES, pH7.5 , 5 millimolar (mM) DTT , 10 % (v/v) glycerol ) at 4C overnight
15m
The next day precipitation will be expected. Transfer the dialyzed material to 50 mL tubes and centrifuge 20000 x g, 4°C, 00:10:00 . The supernatant should be clear. Once again determine the concentration by measurement at A280.
We have never required to concentrate our TEV protease. We make aliquots that correspond to 1-2 mg per tube. Aliquots are flash frozen under liquid nitrogen and then store indefinitely at -80 °C
Our final yield is typically in the 10s of mg/L (on average 50 mg/L)
Expected result
Final sample should look like this
Final sample loaded on a gel
30m
Use of 10xHis-SuperTEV Protease
Use of 10xHis-SuperTEV Protease
We have tested cleavage of substrates containing the TEV protease site of ENLYFQ/GS. We typically use the protease in a 1:50-1:100 (protease:protein mass ratio). However we have used it also at significantly higher ratios. The protease performs well during dialysis in PBS or HBS buffers atRoom temperature or 4 °C overnight
Expected result
His-MBP-Cas9 was purified from Addgene #69090 and cleaved with SuperTEV in a 1:100 ratio. (100 mg total crude protein from elution : 1.4 mg SuperTEV) overnight at 4C
Sample cleavage with SuperTEV protease