TITLE: Purification protocol of Mouse (Mus Musculus) E1-like enzyme ATG7
AUTHORS: Dorotea  Fracchiolla

[DESCRIPTION]
This protocol outlines the procedures for expression and purification of Mouse (Mus Musculus) E1-like enzyme ATG7 (AuTophaGy-related protein) of the ATG8 ubiquitin-like conjugation system in autophagy.

[GUIDELINES]
General information: expression system: Sf9 cells, medium: SF921+Pen/Strep; plasmid origin: Gammoh Noor (University of Edinburgh); internal lab Construct Database number: SMC911; backbone: pFast-BacHT(B); resistance: Ampicillin/Gentamicin; insert: Mus musculus ATG7, Isoform 2_NP_00124647.1; tags & cleavage sites: N-term 6xHis, followed by Tobacco Etch Virus (TEV) cleavage site, 6xHis-TEVcs-mATG7; protein length without tags: 698 aa; Epsilon (all Cysteines reduced, with tags) = 88810 M-1 cm-1 ; MW with tags= 83 kDa; ORF (only mATG7): 

MGDPGLAKLQFAPFNSALDVGFWHELTQKKLNEYRLDEAPKDIKGYYYNGDSAGLPTRLTLEFSAFDMSA STPAHCCPAMGTLHNTNTLEAFKTADKKLLLEQSANEIWEAIKSGAALENPMLLNKFLLLTFADLKKYHF YYWFCCPALCLPESIPLIRGPVSLDQRLSPKQIQALEHAYDDLCRAEGVTALPYFLFKYDDDTVLVSLLK HYSDFFQGQRTKITVGVYDPCNLAQYPGWPLRNFLVLAAHRWSGSFQSVEVLCFRDRTMQGARDVTHSII FEVKLPEMAFSPDCPKAVGWEKNQKGGMGPRMVNLSGCMDPKRLAESSVDLNLKLMCWRLVPTLDLDKVV SVKCLLLGAGTLGCNVARTLMGWGVRHVTFVDNAKISYSNPVRQPLYEFEDCLGGGKPKALAAAERLQKI FPGVNARGFNMSIPMPGHPVNFSDVTMEQARRDVEQLEQLIDNHDVIFLLMDTRESRWLPTVIAASKRKL VINAALGFDTFVVMRHGLKKPKQQGAGDLCPSHLVAPADLGSSLFANIPGYKLGCYFCNDVVAPGDSTRD RTLDQQCTVSRPGLAVIAGALAVELMVSVLQHPEGGYAIASSSDDRMNEPPTSLGLVPHQIRGFLSRFDN VLPVSLAFDKCTACSPKVLDQYEREGFTFLAKVFNSSHSFLEDLTGLTLLHQETQAAEIWDMSDEETV


  
  
  


[STEPS]
SECTION: Infection/expression/harvest
1. Infect 1 L of Sf9 cells growing in Sf921 medium with Penicillin/Streptomycin at 1-1.5 mil/ml cells/volume at 99-100% viability in log phase with Virus 1 (V1) according to viral titer. 
(NOTE: protein yield from 1 lt culture is high, e.i. 32 mg/1lt culture).

SECTION: Infection/expression/harvest
2. Monitor infection and harvest cells when viability goes to 97-98%.
 

SECTION: Protein Purification
3. 
 

SECTION: Protein Purification
4. Resuspend cell pellet corresponding to 1 L  in 50 mL; gently stir at 4 °C until pellet dissolves avoiding bubbling.

SECTION: Protein Purification
5. Additionally, mechanically lyse the cells passing them through a pre-cooled douncer for 3x (10x pestle A followed by 10x pestle B).
SECTION: Protein Purification
6. Clear the lysate by spinning it in a Backman centrifuge, at 25K using a Ti45 Rotor for 45 min at 4 °C.

SECTION: Protein Purification
7. Inject the supernatant onto a 5ml HT column pre-equilibrated with Buffer A at 1ml/min flow rate to allow protein binding.
SECTION: Protein Purification
8. Wash the column for 5CV with Buffer A at 2 ml/min flow rate to remove unspecific bound proteins.
SECTION: Protein Purification
9. Elute protein through a step elution gradient in 50mM, 75mM, 100mM, 150mM, 200mM and 300mM Imidazole concentration. Perform the elution at 1ml/min flow rate.
SECTION: Protein Purification
10. Collect a sample from peak fractions of each elution step and check them on a SDS-PAGE (see gel in the "Guidelines" section). Pool and concentrate fractions corresponding to the peak containing the protein of interest (usually 200mM and/or 300mM Imid.) by spinning them down at4 °C using a 30 kDa cut-off Amicon Filter to 2 ml in a 5810R centrifuge (Eppendorf).  
 


SECTION: Protein Purification
11. Inject 2 mL onto a S200_16/60 column at 44 °C  pre-equilibrated in buffer containing 25mM Hepes pH=7.5, 150mM NaCl and 1mM DTT (see profile in the "Guidelines" section).

SECTION: Protein Purification
12. Check fractions on a 10% SDS-PAGE (see gel in the "Guidelines" section), pool and concentrate down those containing the protein of interest by spinning them at 4 °C using a 30kDa cut-off Amicon Filter in a 5810R centrifuge (Eppendorf). Protein elutes at around 72.5ml, a retention volume corresponding to that of a dimeric globular protein of ~150-160 kDa. 
 


SECTION: Protein Purification
13. Measure protein absorbance at A280 is measured with Spectrophotometer against Size Exclusion Chromatography  buffer.
SECTION: Protein Purification
14. Aliquot the protein, snap freeze it in liquid Nitrogen and store it at -80 °C.