Jan 04, 2023

Public workspaceRecombinant protein expression and purification of fuGFP

DOI
dx.doi.org/10.17504/protocols.io.e6nvwje79lmk/v1
  • 1Millennium Institute for Integrative Biology (iBio), Santiago, Chile;
  • 2Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile
Cesar A Ramirez-Sarmiento
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwje79lmk/v1
Protocol Citation: Javiera A Avilés, Tamara Matute, Isaac Núñez, Maira Rivera, Javiera Reyes, Anibal Arce Medina, Cesar A Ramirez-Sarmiento, Fernan Federici 2023. Recombinant protein expression and purification of fuGFP. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwje79lmk/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: January 04, 2023
Last Modified: January 04, 2023
Protocol Integer ID: 74738
Keywords: RT-LAMP, isothermal amplification, COVID-19, SARS-CoV-2,
Funders Acknowledgements:
ANID Millennium Science Initiative Program
Grant ID: ICN17_022
ANID CONCYTEC
Grant ID: covbio0012
Abstract
This protocol has been optimized for the recombinant expression of fuGFP encoded in an open pTi vector. The plasmid encoding fuGFP used here can be found on reclone.org. The purified protein can be used for teaching about the properties of fluorescent proteins.


Materials
MATERIALS

ReagentSodium phosphate monobasic monohydrateSigma AldrichCatalog #S9638 ReagentPMSFSigma AldrichCatalog #P7626
ReagentSodium phosphate dibasicSigma AldrichCatalog #7558-79-4
ReagentImidazoleSigmaCatalog #I5513
ReagentNaClSigma AldrichCatalog #53014
ReagentHisTrap FF Crude ColumnGe HealthcareCatalog #17528601
ReagentLysozymeThermo Fisher ScientificCatalog #89833

Buffer A, pH 8.0
Concentration50 millimolar (mM) NaPO4, pH 8.0
Concentration300 millimolar (mM) NaCl
Concentration30 millimolar (mM) Imidazole, pH 8.0

Buffer B, pH 8.0
Concentration25 millimolar (mM) Tris-HCl, pH 8.0
Concentration200 millimolar (mM) NaCl
Concentration30 millimolar (mM) Imidazole, pH 8.0

Buffer C, pH 8.0
Concentration25 millimolar (mM) Tris-HCl, pH 8.0
Concentration100 millimolar (mM) NaCl
Concentration300 millimolar (mM) Imidazole, pH 8.0
DAY 1 – Plasmid transformation
DAY 1 – Plasmid transformation
1d
1d
Transform Amount100 ng of the open pTi plasmid containing fuGFP into E. coli BL21 (DE3) competent cells using either heat shock or electroporation.
2h
Spread transformed cells in LB Agar plates supplemented with Concentration0.05 mg/mL Kan. Grow plate overnight at Temperature37 °C .
12h
DAY 2 – Preinoculum
DAY 2 – Preinoculum
1d
1d
Select a single colony from the LB agar plate to prepare a preinoculum in Amount10 mL LB media supplemented with Concentration0.05 mg/mL Kan. Grow overnight at Shaker250 rpm, 37°C .

1d
DAY 3 – Protein Overexpression
DAY 3 – Protein Overexpression
1d
1d
Use the full volume of the preinoculum to inoculate Amount1 L of LB media supplemented withConcentration0.05 mg/mL Kan (1% inoculation). Grow at Shaker200 rpm, 37°C until reaching an optical density at 600 nm (OD600) = 0.8.
4h
Upon reaching OD600 = 0.8, add IPTG to a final concentration of Concentration0.5 millimolar (mM) and incubate overnight at Shaker180 rpm, 18°C

16h
DAY 4 – Protein Purification by IMAC
DAY 4 – Protein Purification by IMAC
6h
6h
Centrifuge the cell culture Centrifigation4000 x g, 4°C, 00:20:00 .Then, resuspend the cell pellet in Amount40 mL of Buffer A freshly supplemented with Concentration0.5 millimolar (mM) PMSF and Concentration0.2 mg/mL lysozyme.

20m
Incubate the resuspended cells Shaker80 rpm, Room temperature , 00:30:00 .
30m
Sonicate on ice for Duration00:08:00 using cycles of Duration00:00:01 ON and Duration00:00:01 OFF at 40% amplitude (Qsonica Q125, 125W).
8m 2s
Centrifuge the unclarified lysate Centrifigation20000 x g, 4°C, 00:20:00 and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
20m
On a 1 mL HisTrap column (GE Healthcare) pre-equilibrated with 10 column volumes (c.v.) (here, 10 mL) of Buffer A, load the supernant. Wash with 20 c.v. of Buffer B. Then, elute with 5 c.v. of Buffer C, collecting the eluted fractions every Amount1 mL in 1.5 ml tubes.
1h
To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with Amount40 µL of 5X Bradford reagent and Amount160 µL of distilled water. Then, add Amount10 µL of each protein fraction and compare against a blank reference sample corresponding to Amount10 µL of Buffer C. You can determine your protein-containing fractions either by absorbance at 595 nm on a plate reader or visually by comparing the blue coloration of each fraction against the blank reference. Pool your fractions and collect a Amount10 µL sample for SDS-PAGE.
5m
For storage, we suggest to do a dialysis against Buffer A, and store at 4º C.
IMAC SDS-PAGE Result
IMAC SDS-PAGE Result
Eluted fractions from immobilized metal affinity chromatography (IMAC) after recombinant protein purification of fuGFP using open pTi vector.
Pooled fractions from immobilized metal affinity chromatography (IMAC) after recombinant protein purification of fuGFP using open pTi vector under blue light.