Expression and purification protocol of the Human (Homo sapiens) LC3B Ubiquitin-like modifier
Dorotea Fracchiolla, Liv Jensen
expression
purification
recombinant
Microtubule-associated proteins 1A/1B light chain 3B
LC3B
mCherry-tag
ASAPCRN
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Abstract
This protocol describes expression and purification procedures for obtaining mCherry-tagged human recombinant Ubiquitin-like modifier LC3B (MAP1LC3B, Microtubule-associated proteins 1A/1B light chain 3B) lacking five C-terminal
amino acids to allow in vitro protein conjugation to target PE, Phosphatidyl Ethanolamine.
Before start
General information
Insert: Homo sapiens LC3B, NP_073729.1; Expression system: E.Coli Rosetta pLyss; plasmid origin: Sascha Martens Lab, Addgene 169168, lab internal construct database number SMC948; backbone: pET-Duet1; plasmid resistance: Ampicillin; tags & cleavage sites: N-term 6xHis, followed by Tobacco Etch Virus (TEV) cleavage site, mCherry tag, LC3B ORF. Ext coeff: 41830 M-1 cm-1 , MW 47,89 kDa.
Steps
Protein Expression
Transform plasmid DNA (Addgene_190237) into E.Coli Rosetta pLyss cells and plate on Ampicillin LB agar plate for at 37°C.
The following day, inoculate a 5mL with 1-2 colonies and grow at 37°C shaking.
The following day, use 5mL to inoculate 1L at 37°C until an OD600(Optical Density) of 0.6 is reached.
Induce protein expression with 400micromolar (µM) and grow for a further 16h 0m 0s at 18°C shaking.
Pellet cells at 4000rpm,4°C in a Sorvall RC6+ centrifuge (Thermo Scientific), discard supernatant and resuspend pellets in ice cold lysis buffer (25 ml/1 lt culture).
Flash freeze resuspended pellets in liquid nitrogen and store at -80°C until purification.
Protein Purification
Perform His-Trap affinity purification followed by Size Exclusion Chromatography.
Purification of LC3B Ubiquitin-like modifier
Cells are lised via freeze/thaw cycles and sonication: thaw pellet corresponding to 1L culture by freeze/thawing in Room temperature water bath. All following steps are to be executed at 4°C or 4On ice.
Lyse cells by sonicating them with an immersion Tip Sonicator ( 2x 0h 0m 30s). Note: adjust times and intensity according to the available instrument.
Clear lysate by spinning in a Beckman centrifuge, at 40000x g,4°C.
Inject soluble fraction onto a 5ml HT column operating at 4°C pre-equilibrated in Buffer A at 1ml/min flow rate.
Wash column for 5 at 2 ml/min flow rate to remove unspecific bound proteins.
Elute protein at 300mM Imidazole concentration. Perform elution at 1ml/min flow rate.
Perform TEV cleavage with 1mg/ml TEV protease overnight at 4˚C while dialyzing against elution buffer containing no imidazole.
Apply cleavage reaction to equilibrated HT column, and collect the flow-through.
Measure protein absorbance at A280 with a Spectrophotometer against dialysis buffer.
Aliquot protein, flash freeze in liquid Nitrogen and store at -80°C.
