TITLE: Small Scale purification test for expression of human PINK1 in insect cells
AUTHORS: Verena Dederer, Olawale G. Raimi, Miratul M K Muqit, Stefan Knapp, Sebastian Mathea

[DESCRIPTION]
Purified, recombinant proteins are essential for biochemical characterization and structural studies. However, every protein is unique in its in vitro behaviour and thus, establishing a stable expression system and purification protocol can be challenging. Therefore, testing several constructs in parallel may increase the chances of getting a high quality protein prep while minimizing the laborious work. In this protocol we are providing an overview of the main steps in order to clone and test several constructs with varying N-and C-terminal boundaries for expression of human PTEN induced kinase 1(PINK1) in insect cells (this protocol is adapted from 1). PINK1, together with its counterpart Parkin, recognize damaged mitochondria and provoke their degradation by mitophagy, a mitochondrial quality control pathway (for review see 2). Mutations of PINK1 are associated with early-onset Parkinson’s disease. Therefore, understanding its structure will help in unravelling its cellular function and hopefully will lead to development of new treatment strategies.

[STEPS]
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
1. Amplify region of interest from human PINK1 gene (OHu25380D; Genscript) with primers
endcoding 5’TACTTCCAATCCATG extension for forward and 5’TATCCACCTTTACTGTCA extension for reverse primers.
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
2. DpnI digest template DNA.
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
3. Purify PCR product using MultiScreen 96well PCR clean-up (Merck Millipore, MSNU03010).
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
4. Treat purified PCR products with T4 polymerase (NEB #M0203L) in presence of dCTP.
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
5. Linearize and purify pFB-6HZB using BseRI (NEB# R0581L).
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
6. Treat linearized vector with T4 polymerase in presence of dGTP to generate complementary sequence overhangs.
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
7. Anneal vector and insert (ratio 1:4) in 10 µL at 4 Room temperature for 60 min.

SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
8. Transform annealing mix into E. coli. 
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
9. Plate transformants onto LB agar plates containing 100 µg/mL ampicillin and 5 % sucrose for nick repair and selection.
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
10. Inoculate liquid culture of successfully growing transformants for plasmid isolation.
SECTION: Section 1: Cloning of human PINK1 constructs into pFB-6HZB
11. Validate successful cloning by PCR or sequencing.
SECTION: Section 2: Baculovirus generation and small-scale test expression
12. Generate bacmids by transforming the plasmids into DH10bac cells.
SECTION: Section 2: Baculovirus generation and small-scale test expression
14. Pick white colonies and inoculate overnight culture for bacmid isolation.


SECTION: Section 2: Baculovirus generation and small-scale test expression
16. Produce recombinant baculovirus by transfecting bacmids into pre-seeded insect cells: 2 mL 0.2 x10^6 cells/mL in 24 well plate.
SECTION: Section 2: Baculovirus generation and small-scale test expression
17. Prepare transfection mix (per well): 1 µL   diluted in 49 µL medium and 4 µL   diluted in 46 µL 

SECTION: Section 2: Baculovirus generation and small-scale test expression
18. Incubate transfection mix for 15 min at Room temperature

SECTION: Section 2: Baculovirus generation and small-scale test expression
13. Select for positive transformants by plating cells onto LB agar plates containing 50μg/mL kanamycin, 7μg/mL gentamycin, 10μg/mL tetracycline, 100μg/mL Bluo-gal, and 40μg/mL IPTG.


SECTION: Section 2: Baculovirus generation and small-scale test expression
15. Isolate bacmids by alkine lysis and genomic DNA precipitation using sodium acetate precipitation followed by isopropanol wash.


SECTION: Section 2: Baculovirus generation and small-scale test expression
19. Add transfection mix to the cells
SECTION: Section 2: Baculovirus generation and small-scale test expression
20. Incubate cells for 10080 min at 27 °C for virus production

SECTION: Section 2: Baculovirus generation and small-scale test expression
21. Transfer virus containing supernatant to fresh plate.
SECTION: Section 2: Baculovirus generation and small-scale test expression
22. For protein expression seed 3 mL insect cells (2x10^6 cells/mL) medium in 24 deep-well block.

SECTION: Section 2: Baculovirus generation and small-scale test expression
23. Infect cells with prepared virus containing supernatant (MOI>2).
SECTION: Section 2: Baculovirus generation and small-scale test expression
24. Incubate cells shaking    

SECTION: Section 2: Baculovirus generation and small-scale test expression
25. Harvest the cells by centrifugation with 1000 x g, 20 min, 4 °C .

SECTION: Section 2: Baculovirus generation and small-scale test expression
26. Freeze cell pellets until further use.
SECTION: Section 3: Test purification
27. Resuspend cell pellets in 2 mL pH 7.4    

SECTION: Section 3: Test purification
28. Lyse cells by sonication (24-tip horn; sonication parameters: 35% amplitude, 5 s pulse / 10 s pause, 3 min total pulse time)
SECTION: Section 3: Test purification
29. Clear lysate by centrifugation 13000 rpm, 30 min, 4 °C

 

SECTION: Section 3: Test purification
30. Load cleared lysate onto 25 µL pre-equilibrate Ni-Sepharose beads in gravity flow columns

SECTION: Section 3: Test purification
31. Wash beads with 2 mL pH 7.4 

SECTION: Section 3: Test purification
32. Elute His6-Z-PINK1 constructs with 50 µL lysis buffer containing 300 Molarity (M) imidazole
 

SECTION: Section 3: Test purification
33. Perform SDS-PAGE analysis and stain gel with Coomassie stain or further proceed with Western
blot analysis.