Jun 02, 2026
Expression, purification, and characterization of CtVPS13(1-635) with calmodulin
- Dazhi Li1,2,
- Karin Reinisch1,2
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Dazhi Li, Karin Reinisch 2026. Expression, purification, and characterization of CtVPS13(1-635) with calmodulin. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11m17vr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 19, 2025
Last Modified: June 02, 2026
Protocol Integer ID: 230221
Keywords: Lipid transport protein, VPS13, Calmodulin, vps13 from chaetomium thermophilum, lysosome contact site protein, calmodulin vps13c, important for lysosomal membrane damage repair, protein, organellar membrane, folding of vps13 family, lysosomal membrane damage repair, significance of calmodulin, chaetomium thermophilum, membrane, like protein, vps13c, vps13 family, calmodulin, vps13, uncovered novel regulatory mechanism, purification, novel regulatory mechanism, lipid, bacteria
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: ASAP-000580
NIH
Grant ID: R35GM131715
Abstract
VPS13C is a ER-lysosome contact site protein that is thought to transport lipids between the two organellar membranes and important for lysosomal membrane damage repair. We employed structure-function analysis of purified VPS13C and uncovered novel regulatory mechanisms. This protocol describes the purification of a fragment of VPS13 from Chaetomium thermophilum (Ct) from bacteria, which can only be achieved when co-expressed with calmodulin (or likely, calmodulin-like proteins). This suggests the significance of calmodulin (or calmodulin-like protein) to the folding of VPS13 family of proteins.
Materials
BL21(De3) pLys cells (Cat. #200132, Agilent)
IPTG (Cat. #I56000, RPI)
imidazole (Cat. #56750, Sigma Aldrich)
Talon metal affinity Resin (Cat. #635502, Takara)
Protein Expression in BL21
6h
Transform pETDuet-6xHis-Strep-CtVPS13(1–635) or pETDuet-6xHis-Strep-CtVPS13(1–635)-6xHis-HsCaM into homemade BL21(DE3) pLys cells.
Note that the later construct has VPS13 fragment in the first ORF and CaM in the second ORF.
1h
Grow 1 L LB culture to OD600 = 0.8, induce with 0.5 mM IPTG, and continue expression at 18 °C for 16:00:00 .
4h
Harvest cells and resuspend in 20 mL buffer A (500 mM NaCl, 50 mM HEPES, pH 7.8, 10% glycerol, 1 mM TCEP) supplemented with 1× protease inhibitor cocktail and 5 mM imidazole.
1h
Protein Purification
10h
Lyse cells by passing the suspension five times through an Avestin EmulsiFlex-C5 at 7,500 psi.
30m
Clarify the lysate by centrifugation at 36,945 × g for 00:30:00 at 4 °C in a JA-20 rotor.
30m
Incubate the supernatant with 100 μL Talon metal affinity resin for00:30:00 at 4 °C with rotation.
30m
Wash the resin with 50 column volumes of buffer A containing 20 mM imidazole.
30m
Elute the protein with buffer A containing 300 mM imidazole in 1 mL fractions, incubating the resin for 00:05:00 during each elution.
30m
Optional: Tandem Strep purification to remove free calmodulin. Because both the His-tagged CtVPS13 fragment and His-tagged free calmodulin were captured by Talon resin.
4h
Incubate the Talon eluate with 50 μL Strep-Tactin XT 4Flow high-capacity resin for 02:00:00 at 4 °C .
2h
Wash the resin three times with 15 mL buffer A.
30m
Elute bound protein three times with 50 μL buffer A supplemented with 50 mM D-biotin, incubating for 00:20:00 for each elution.
1h
Protein Characterization
6h
Analyze the eluates on SDS-PAGE. The CtVPS13 fragment is only soluble and purifiable in the presence of calmodulin.
2h
Analyze the purified protein by size-exclusion chromatography (SEC) on a Superose 6 Increase column equilibrated with buffer A.
1h
Analyze the fractions from SEC on SDS-PAGE. The CtVPS13 fragment forms a 1:1 complex with calmodulin and elutes as a peak consistent with a dimeric complex.
2h
Analyze the purified protein by negative-stain electron microscopy. 2D classification of picked particles in CryoSPARC revealed homogeneous class averages, consistent with a tube-like dimeric assembly of the CtVPS13 fragment predicted by AlphaFold.
1h