TITLE: Insect cell expression of wildtype and variant LRRK1full length protein 
AUTHORS: Deep Chatterjee, Sebastian Mathea, Stefan Knapp

[DESCRIPTION]
The production of LRRK1 protein and its variants was performed using bacculovirus expression system. The method includes three major steps cloning, large scale expression and protein purification. The high quality LRRK1 protein produced through this method has been used for further biochemical and biophysical studies.

[GUIDELINES]
Expression 
Protein quality depends on several factors, for eg. condition of insect cells, virus quality, expression time etc.

Purification
Every steps are performed at 4 °C 
Column and beads has to properly equilibrated or regenerated before starting

Protein stability during purification can be improved by using different strategies, such as increasing salt concentration, use of additives or protease inhibitors


[STEPS]
SECTION: Cloning &amp; mutagenesis
1. Amplify the DNA coding for the human LRRK1 residues 20 to 2015 (OHu72031 from Genscript) using the forward primer TACTTCCAATCCGCTGTGTGTCCAGAACGTGCCATGG and the reverse primer TATCCACCTTTACTGTCACCTTCTCTTGCGAGTGCAAGCCTCC. PCR was performed by applying a touch-down protocol.

Thermal cycling procedure:

95 °C 10 min 
(95 °C 30 s 68 °C 30 s 68 °C 10 min ) X 5
(95 °C 30 s 60 °C 30 s 68 °C 10 min ) X 5
(95 °C 30 s 55 °C 30 s 68 °C 10 min ) X 5
(95 °C 30 s 50 °C 30 s 68 °C 10 min ) X 20
68 °C 10 min

SECTION: Expression
5. 9.6 L of exponentially growing Sf9 cells (2 X 106 cells/mL in Lonza Insect-XPRESS medium) were infected with high-titre Baculovirus suspension. 


SECTION: Purification
7. The pelletes were washed with PBS, re-suspended in lysis buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% (by vol) glycerol). 


SECTION: Cloning &amp; mutagenesis
2. 
The T4 polymerase-treated amplicon was inserted into the transfer vector pFB-6HZB (SGC) by ligation-independent cloning. 
SECTION: Cloning &amp; mutagenesis
3. 
Point mutations were introduced applying the QuikChange method. 
SECTION: Cloning &amp; mutagenesis
4. The resulting plasmids were utilized for the generation of recombinant Baculoviruses according to the Bac-to-Bac expression system protocol (Invitrogen). 
SECTION: Expression
6. After 3960 min of incubation (27 °C and 90 rpm), cells were harvested by centrifugation. 

SECTION: Purification
8.  Lysed by sonication on ice using a 13‑mm probe (35% amplitude, 5 s pulse / 10 s pause, 3 min total pulse time).
SECTION: Purification
9. The lysate was cleared by centrifugation (60 min, 100.000 x g, 4 °C).

SECTION: Purification
10. The supernatant is loaded onto a previously equilibrated Ni-beads (Cytiva #17531803) in gravity flow columns.
SECTION: Purification
11. After vigorous washing (20-30 CV) with lysis buffer, the His6-Z tagged protein was eluted in lysis buffer containing imidazole (50 mM HEPES pH 7.4 500 mM NaCl, 300 mM imidazole, 0.5 mM TCEP, 5% glycerol). 

SECTION: Purification
12. 
The eluate was diluted with buffer containing no NaCl, (20 mM HEPES pH 7.4 0.5 mM TCEP, 5% glycerol) in order to reduce the NaCl-concentration to 250 mM 

SECTION: Purification
15. His6 Z TEV-LRRK1 was eluted with a 250 mM to 2.5 M NaCl gradient. 
SECTION: Purification
16. The protein was treated with TEV protease (molar ratio TEV to substrate about 1:50) overnight to cleave the His6 Z tag. 
SECTION: Purification
17. Contaminating proteins, the cleaved tag, uncleaved protein and TEV protease were removed by another combined SP-Sepharose Ni NTA step. 
SECTION: Purification
18. LRRK1 was concentrated and subjected to gel filtration in storage buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 0.5% glycerol) using an AKTA Pure system combined with an S200 gel filtration column. 

SECTION: Purification
19. The final yield as calculated from UV absorbance was 0.1 mg/L.
SECTION: Purification
13. The diluted protein was loaded onto an 5 mL SP-Sepharose column (Cytiva #17505701) connected to an ÄKTA Basic system. 
SECTION: Purification
14. Wash with buffer containing 250 mM NaCl (20 mM HEPES pH 7.4, 250 mM NaCl, 0.5 mM TCEP, 5% glycerol) until the absorbance of the flowthrough is below 5 mAU