Recombinant Protein Purification (GST-tag, B-PER)
Aim
Quick extraction and purification of GST-tagged recombinant protein expressed in E.coli
Materials
BL21 (C+ or +pLysl or Rossetta)
LB liquid (+ampicilin +Chloramphenicol)
0.3M IPTG (in 50% EtOH, store at -20℃)
B-PER (w or w/o apropriate NaCl, b-ME or etc)
Glutathione Sepharose 4B
L-reduced glutathione
PBS (or apropiate elutin buffer)
Wash Buffer
50 mM Tris-HCl pH7.0
0.01% NP-40
Elution Buffer
50 mM Tris-HCl pH7.0
0.01% NP-40
20 mM L-reduced glutathione (pH should be adjusted to 7-8 by 10N NaOH)
Methods
Day0
transformation pGEX plasmid to BL21.
Direct liquid culture in 2 ml LB+amp+chloram. (Or streak on the plate and pick up single colony)
shake at 37℃ O/N.
Day1(Protein expression)
Pre-culture is diluted into 1:100 in 50-200 ml LB+amp+chloram.
37℃ 1.5-2.5 h shake, to OD600=0.4-0.6 (growth speed and optimal OD depend on your protein).
Cool down culture to 25℃ or 18℃.
add IPTG final conc.0.3 mM (optimal concentration depends on your protein)
shake 25℃ for 4-8 h, or 18℃ for 12-24 h.
Cool down culture to 4℃ on ice.
collect E.coli by centrifugation at 5000 rpm.
Discard medium and go to purification step, or you can store it at -80℃.
Day2 (purification)
add apropriate amout of B-PER (5 ml B-PER for 50 ml culture), and suspend pellet by pipetting.
(option: DNase and Lysozyme can improve the lysis)
rotate at R.T for 10 min.
transfer sample to 2 ml tube and 10k rpm at 4℃ for 10-30 min.
take sup into new 5 ml tube and add neutrized 250 ul GS4B.
rotate at 4℃ for 1h.
wash with wash buffer 5 ml 3 times.
add 250 ul Elution Buffer
take sup
repeat elution 3~5 times
Remarks
pLysl or additional rare codons are kept in E.coli by Chloramphenicol selection.
Each parameters should be optimized for your protein.
You should collect samples in each purification step (E.coli IPTG +/-, Cell lysate, beads, and flowthrough) .
Date : 2018.8.30
Modified Date :
Author :y-goto.icon