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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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 </div><div class = "text-block">amino acids to allow in vitro protein conjugation to target PE, Phosphatidyl Ethanolamine.</div></div>

[STEPS]
?. [Protein Expression]
Transform plasmid DNA (Addgene 169168 , SMC948) into E.Coli Rosetta pLyss cells and plate on Ampicillin/Chloramphenicol LB agar plate for   at .
37 °C
?. [Protein Expression]
The following day, inoculate a  with 1-2 colonies and grow  at  shaking.
[LB + Amp/Cam pre-culture ]
37 °C
?. [Protein Expression]
The following day, use  to inoculate  at  until an OD600 (Optical Density) of 0.4 is reached.
[pre-culture]
[LB medium + Amp/Cam]
37 °C
?. [Protein Expression]
Cool down the culture to  and grow to an OD600 = 0.8.
18 °C
?. [Protein Expression]
Pellet cells at  in a Sorvall RC6+ centrifuge (Thermo Scientific), discard supernatant and resuspend pellets in ice cold lysis buffer (25 ml/1 lt culture).
Centrifuge: 4000 33, 15 min, 4 10
?. [Protein Purification]
Perform His-Trap affinity purification followed by Size Exclusion Chromatography.
?. [Purification of LC3B Ubiquitin-like modifier]
Lyse cells by sonicating them with an immersion Tip Sonicator (2x ). Note: adjust times and intensity according to the available instrument.
?. [Purification of LC3B Ubiquitin-like modifier]
Filter supernatant through a 0.45 µm filter and inject onto a 5ml HT column operating at   pre-equilibrated in Buffer A at 1ml/min flow rate.
4 °C
?. [Purification of LC3B Ubiquitin-like modifier]
Wash column for  at 2 ml/min flow rate to remove unspecific bound proteins.
[with Buffer A]
?. [Purification of LC3B Ubiquitin-like modifier]
Elute protein through a step elution gradient in 50mM, 75mM, 100mM, 150mM, 200mM and 300mM Imidazole concentration. Perform elution at 1ml/min flow rate. Collect peak fractions at each step.
?. [Purification of LC3B Ubiquitin-like modifier]
Check peak fractions of each step on a SDS-PAGE (see gel below). Pool and concentrate those containing the protein of interest (75 and 100mM Imidazole step) by spinning at  down in an Amicon Filter 30kDa cut-off to .
4 °C
[final volume]
Centrifugation steps are kept short ( ) to avoid protein local concentration/aggregation on the filter.
?. [Purification of LC3B Ubiquitin-like modifier]
Cells are lised via freeze/thaw cycles and sonication: thaw pellet corresponding to  culture by freeze/thawing in  water bath. All following steps are to be executed at  or .
1 L
0 Room temperature
4 °C
on ice
?. [Protein Expression]
Induce protein expression with  and grow for a further  at  shaking.
[IPTG]
18 °C
?. [Protein Expression]
Flash freeze resuspended pellets in liquid nitrogen and store at  until purification.
-80 °C
?. [Purification of LC3B Ubiquitin-like modifier]
Clear lysate by spinning in a Beckman centrifuge, at .
Centrifuge: 40000 34, 30 min, 4 10
?. [Purification of LC3B Ubiquitin-like modifier]
Inject  onto a S75_16/60 column pre-equilibrated in SEC buffer.
[protein]
?. [Purification of LC3B Ubiquitin-like modifier]
Check fractions on a 10% SDS-PAGE (see gel below). Pool and concentrate those containing the proteins of interest down at  in an Amicon Filter 30kDa cut-off.
4 °C
Centrifugation steps are kept short ( ) to avoid protein local concentration/aggregation on the filter.
?. [Purification of LC3B Ubiquitin-like modifier]
Measure protein absorbance at A280 with a Spectrophotometer against Size Exclusion Chromatography purification buffer (MW = 47,89 kDa ; Extinction coefficient = 41830 M-1cm-1).
?. [Purification of LC3B Ubiquitin-like modifier]
Aliquot protein, flash freeze in liquid Nitrogen and store at .
-80 °C