Jan 04, 2023

Recombinant protein expression and purification of fuGFP

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwje79lmk/v1
Recombinant protein expression and purification of fuGFP
  • 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: https://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, recombinant expression of fugfp, encoding fugfp, fugfp, fugfp this protocol, fluorescent protein, recombinant protein expression, properties of fluorescent protein, purified protein, protein, recombinant expression, purification, plasmid, open pti vector, purification of fugfp,
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

Sodium phosphate monobasic monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9638 PMSFMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #7558-79-4
ImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
HisTrap FF Crude ColumnGE HealthcareCatalog #17528601
LysozymeThermo Fisher ScientificCatalog #89833

Buffer A, pH 8.0
50 millimolar (mM) NaPO4, pH 8.0
300 millimolar (mM) NaCl
30 millimolar (mM) Imidazole, pH 8.0

Buffer B, pH 8.0
25 millimolar (mM) Tris-HCl, pH 8.0
200 millimolar (mM) NaCl
30 millimolar (mM) Imidazole, pH 8.0

Buffer C, pH 8.0
25 millimolar (mM) Tris-HCl, pH 8.0
100 millimolar (mM) NaCl
300 millimolar (mM) Imidazole, pH 8.0
DAY 1 – Plasmid transformation
1d
Transform 100 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 0.05 mg/mL Kan. Grow plate overnight at 37 °C .
12h
DAY 2 – Preinoculum
1d
Select a single colony from the LB agar plate to prepare a preinoculum in 10 mL LB media supplemented with 0.05 mg/mL Kan. Grow overnight at 250 rpm, 37°C .

1d
DAY 3 – Protein Overexpression
1d
Use the full volume of the preinoculum to inoculate 1 L of LB media supplemented with0.05 mg/mL Kan (1% inoculation). Grow at 200 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 0.5 millimolar (mM) and incubate overnight at 180 rpm, 18°C

16h
DAY 4 – Protein Purification by IMAC
6h
Centrifuge the cell culture 4000 x g, 4°C, 00:20:00 .Then, resuspend the cell pellet in 40 mL of Buffer A freshly supplemented with 0.5 millimolar (mM) PMSF and 0.2 mg/mL lysozyme.

20m
Incubate the resuspended cells 80 rpm, Room temperature , 00:30:00 .
30m
Sonicate on ice for 00:08:00 using cycles of 00:00:01 ON and 00:00:01 OFF at 40% amplitude (Qsonica Q125, 125W).
8m 2s
Centrifuge the unclarified lysate 20000 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 1 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 40 µL of 5X Bradford reagent and 160 µL of distilled water. Then, add 10 µL of each protein fraction and compare against a blank reference sample corresponding to 10 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 10 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
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.