TITLE: Expression and purification protocol of the Human (Homo sapiens) LC3B Ubiquitin-like modifier
AUTHORS: Dorotea  Fracchiolla, Liv Jensen

[DESCRIPTION]
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.

[GUIDELINES]
Affinity purification

  
  
Size Exclusion Chromatography

  
  


[STEPS]
SECTION: Protein Expression
1. Transform plasmid DNA (Addgene_190237) into E.Coli Rosetta pLyss cells and plate on Ampicillin LB agar plate for    at 37 °C.

SECTION: Protein Expression
2. The following day, inoculate a 5 mL with 1-2 colonies and grow   at 37 °C shaking. 

SECTION: Protein Expression
3. The following day, use 5 mL to inoculate 1 L at 37 °C until an OD600 (Optical Density) of 0.6 is reached. 

SECTION: Protein Expression
5. Pellet cells at 4000 rpm, 15 min, 4 °C in a Sorvall RC6+ centrifuge (Thermo Scientific), discard supernatant and resuspend pellets in ice cold lysis buffer (25 ml/1 lt culture).
SECTION: Protein Purification
7. Perform His-Trap affinity purification followed by Size Exclusion Chromatography.
SECTION: Purification of LC3B Ubiquitin-like modifier
9. Lyse cells by sonicating them with an immersion Tip Sonicator (2x 30 s). Note: adjust times and intensity according to the available instrument.

SECTION: Purification of LC3B Ubiquitin-like modifier
11. Inject soluble fraction onto a 5ml HT column operating at 4 °C  pre-equilibrated in Buffer A at 1ml/min flow rate. 

SECTION: Purification of LC3B Ubiquitin-like modifier
12. Wash column for 5 1406 at 2 ml/min flow rate to remove unspecific bound proteins.
SECTION: Purification of LC3B Ubiquitin-like modifier
13. Elute protein at 300mM Imidazole concentration. Perform elution at 1ml/min flow rate. 
 

SECTION: Purification of LC3B Ubiquitin-like modifier
8. Cells are lised via freeze/thaw cycles and sonication: thaw pellet corresponding to 1 L culture by freeze/thawing in Room temperature water bath. All following steps are to be executed at 4 °C or on ice.

SECTION: Protein Expression
4. Induce protein expression with 400 micromolar (µM) and grow for a further 960 min at 18 °C shaking. 

SECTION: Protein Expression
6. Flash freeze resuspended pellets in liquid nitrogen and store at -80 °C until purification.

SECTION: Purification of LC3B Ubiquitin-like modifier
10. Clear lysate by spinning in a Beckman centrifuge, at 40000 x g, 30 min, 4 °C.
SECTION: Purification of LC3B Ubiquitin-like modifier
16. Measure protein absorbance at A280 with a Spectrophotometer against dialysis buffer. 
SECTION: Purification of LC3B Ubiquitin-like modifier
17. Aliquot protein, flash freeze in liquid Nitrogen and store at -80 °C.
SECTION: Purification of LC3B Ubiquitin-like modifier
14. Perform TEV cleavage with 1mg/ml TEV protease overnight at 4˚C while dialyzing against elution buffer containing no imidazole.  
SECTION: Purification of LC3B Ubiquitin-like modifier
15. Apply cleavage reaction to equilibrated HT column, and collect the flow-through.