Protein expression and purification
This protocol is extracted from research article:
The structure of the actin filament uncapping complex mediated by twinfilin
Sci Adv, Jan 27, 2021; DOI: 10.1126/sciadv.abd5271

The gene sequences encoding full-length human twinfilin-1 and profilin-1 were codon-optimized for Escherichia coli, synthesized (GenScript), and cloned into a pSY5 vector that includes an N-terminal eight-histidine tag followed by a human rhinovirus 3C protease cleavage site (51). The mouse full-length CP (α1/β2) construct was provided by P. Lappalainen (University of Helsinki, Finland). The construct was in the pRSFDuet-1 vector designed for coexpression of two interacting target proteins and contains an N-terminal six-histidine tag on the α-subunit (52).

All constructs encoding proteins of interest were transformed and recombinantly expressed in phage-resistant E. coli strain BL21 Star (DE3) (New England Biolabs). Fresh LB medium supplemented with either ampicillin (pSY5) or kanamycin (pRSFDuet-1) was inoculated with respective overnight cultures and shaken at 37°C until the cell density reached OD600 (optical density at 600 nm) ~ 0.6. Cells were induced for protein expression with 0.25 mM isopropyl-β-d-thiogalactopyranoside at 16°C overnight. Cells were harvested via centrifugation at 4000g at 4°C for 1 hour, and the pellets were resuspended in 50 ml of His-binding buffer [50 mM tris-HCl (pH 7.5), 500 mM NaCl, 20 mM imidazole, and one protease inhibitor tablet]. The cells were lysed by sonication with a Vibra-Cell ultrasonic processor and clarified by centrifugation at 19,000 rpm in SS-34 rotor using an RC 5C Plus centrifuge (Sorvall) at 4°C for 1 hour followed by filtration through a 0.45-μm Minisart syringe filter (Sartorius). The proteins were purified on an ÄKTAxpress system (GE Healthcare) by affinity chromatography using 5 ml of HisTrap FF column with or without (CP) on-column cleavage of the His-tag with human rhinovirus 3C protease. Proteins purified with the His-tag were eluted in buffer containing 50 mM tris-HCl (pH 7.5), 500 mM NaCl, and 500 mM imidazole. The eluted proteins were concentrated to 5 ml and subjected to size exclusion chromatography (SEC) in a Superdex 75 pg column (GE Healthcare) preequilibrated with buffer containing 50 mM tris-HCl (pH 7.5) and 150 mM NaCl. All purified proteins were verified by SDS–polyacrylamide gel electrophoresis (PAGE) before being snap-frozen and stored at −80°C.

MBP-tagged twinfilin tail constituting residues Gln321-Asp350 was polymerase chain reaction (PCR)–amplified from full-length human twinfilin-1 expression plasmid and inserted into pSY7 vector, which incorporates a cleavable N-terminal histidine and MBP tag. MBP control was prepared by expressing pSY7 vector alone in BL21(DE3) E. coli. Both MBP-tagged twinfilin tail and MBP were purified by affinity chromatography using 1 ml of HisTrap FF column with on-column digestion with human rhinovirus 3C protease to remove the histidine tag. The proteins were further purified by gel filtration chromatography with a Superdex 75 pg column (GE Healthcare) equilibrated with 50 mM tris-HCl (pH 9) and 150 mM NaCl.

Human V-1 (pGEX-6P-3 vector) was expressed as a glutathione S-transferase (GST) fusion E. coli BL21 Star (DE3) and affinity-purified on an ÄKTAxpress system (GE Healthcare) by loading cleared lysate onto 1 ml of GSTrap FF Column (GE Healthcare) equilibrated with 50 mM tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM dithiothreitol (DTT). GST tag was removed by on-column digestion with human rhinovirus 3C protease overnight followed by further purification by gel filtration chromatography. CARMIL CP–binding region (CBR) was expressed and purified as previously described (34).

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