Recombinant protein expression and purification of fuGFP
Maira Rivera, Javiera Reyes, Fernan Federici, Cesar A Ramirez-Sarmiento, Isaac Sir Núñez, Tamara Matute, Anibal Arce Medina, Javiera A Avilés
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.
Steps
DAY 1 – Plasmid transformation
Transform 100ngof the open pTi plasmid containing fuGFP into E. coli BL21 (DE3) competent cells using either heat shock or electroporation.
Spread transformed cells in LB Agar plates supplemented with 0.05mg/mLKan. Grow plate overnight at 37°C.
DAY 2 – Preinoculum
Select a single colony from the LB agar plate to prepare a preinoculum in 10mL LB media supplemented with 0.05mg/mLKan. Grow overnight at 250rpm.
DAY 3 – Protein Overexpression
Use the full volume of the preinoculum to inoculate 1L of LB media supplemented with0.05mg/mLKan (1% inoculation). Grow at 200rpm until reaching an optical density at 600 nm (OD600) = 0.8.
Upon reaching OD600= 0.8, add IPTG to a final concentration of 0.5millimolar (mM) and incubate overnight at 180rpm
DAY 4 – Protein Purification by IMAC
Centrifuge the cell culture 4000x g,4°C.Then, resuspend the cell pellet in 40mL of Buffer A freshly supplemented with 0.5millimolar (mM) PMSF and 0.2mg/mL lysozyme.
Incubate the resuspended cells 80rpm.
Sonicate on ice for 0h 8m 0s using cycles of 0h 0m 1s ON and 0h 0m 1s OFF at 40% amplitude (Qsonica Q125, 125W).
Centrifuge the unclarified lysate 20000x g,4°C and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
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 1mLin 1.5 ml tubes.
To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with 40µLof 5X Bradford reagent and 160µLof distilled water. Then, add 10µLof each protein fraction and compare against a blank reference sample corresponding to 10µLof 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 10µL sample for SDS-PAGE.
For storage, we suggest to do a dialysis against Buffer A , and store at 4º C.
IMAC SDS-PAGE Result
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