Small Scale purification test for expression of human PINK1 in insect cells
Sebastian Mathea, Stefan Knapp, Verena Dederer, Olawale G. Raimi, Miratul M K Muqit
Abstract
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 1: Cloning of human PINK1 constructs into pFB-6HZB
Amplify region of interest from human PINK1 gene (OHu25380D; Genscript) with primers
endcoding 5’TACTTCCAATCCATG extension for forward and 5’TATCCACCTTTACT TCA A extension for reverse primers.
DpnI digest template DNA.
Purify PCR product using MultiScreen 96well PCR clean-up (Merck Millipore, MSNU03010).
Treat purified PCR products with T4 polymerase (NEB #M0203L) in presence of dCTP.
Linearize and purify pFB-6HZB using BseRI (NEB# R0581L).
Treat linearized vector with T4 polymerase in presence of dGTP to generate complementary sequence overhangs.
Anneal vector and insert (ratio 1:4) in 10µL at 4Room temperature for 1h 0m 0s.
Transform annealing mix into E. coli.
Plate transformants onto LB agar plates containing 100 µg/mL ampicillin and 5 % sucrose for nick repair and selection.
Inoculate liquid culture of successfully growing transformants for plasmid isolation.
Validate successful cloning by PCR or sequencing.
Section 2: Baculovirus generation and small-scale test expression
Generate bacmids by transforming the plasmids into DH10bac cells.
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.
Pick white colonies and inoculate overnight culture for bacmid isolation.
Isolate bacmids by alkine lysis and genomic DNA precipitation using sodium acetate precipitation followed by isopropanol wash.
Produce recombinant baculovirus by transfecting bacmids into pre-seeded insect cells: 2mL 0.2 x10^6 cells/mL in 24 well plate.
Prepare transfection mix (per well): 1µL Sample diluted in 49µL medium and 4µL Sample diluted in 46µL``Sample
Incubate transfection mix for 0h 15m 0s at Room temperature
Add transfection mix to the cells
Incubate cells for 168h 0m 0s at 27°C for virus production
Transfer virus containing supernatant to fresh plate.
For protein expression seed 3mL insect cells (2x10^6 cells/mL) medium in 24 deep-well block.
Infect cells with prepared virus containing supernatant (MOI>2).
Incubate cells shaking 180rpm
Harvest the cells by centrifugation with 1000x g,4°C .
Freeze cell pellets until further use.
Section 3: Test purification
Resuspend cell pellets in 2mL 7.4
Lyse cells by sonication (24-tip horn; sonication parameters: 35% amplitude, 5 s pulse / 10 s pause, 3 min total pulse time)
Clear lysate by centrifugation 13000rpm,4°C
Load cleared lysate onto 25µL pre-equilibrate Ni-Sepharose beads in gravity flow columns
Wash beads with 2mL 7.4
Elute His6-Z-PINK1 constructs with 50µL lysis buffer containing 300Molarity (M) imidazole
Perform SDS-PAGE analysis and stain gel with Coomassie stain or further proceed with Western
blot analysis.
