The codon-optimized sequences of PilA1 from strains R20291, NAP08 and CD160, starting with Serine 26, were cloned into a Maltose Binding Fusion vector making use of previously described surface entropy reduction mutations (pMal E) (Moon et al., 2010 (link)). A C-terminal 6xHIS tag was included for ease of purification. These clones were transformed into BL21 (DE3) pLysS cells and grown to saturation overnight with shaking at 37C in LB media with 50ug/ml ampicillin. These saturation cultures were then diluted into fresh LB-ampicillin and grown to and OD of 0.4–0.6 at 37C. These flasks were transferred to a refrigerated orbital shaker and cooled to 18C before induction with 30 μM Isopropyl a-D-1-thiogalactopyranoside (IPTG). These flasks were allowed to grow overnight before being harvested by centrifugation at 7500 x g for ten minutes. The cells were then lysed using lysozyme and the resulting lysate centrifuged again, this time at 20000 x g for 30 minutes. The supernatant was purified using a nickel-NTA column and the elution further purified through size exclusion chromatography over a GE S200 superdex column using an Åkta purifier FPLC.
S 200 superdex column
Manufactured by GE Healthcare
The S-200 Superdex column is a size exclusion chromatography (SEC) column used for the separation and purification of a wide range of biomolecules, including proteins, peptides, nucleic acids, and other macromolecules. The column is packed with a cross-linked agarose-dextran matrix that provides high resolution separation and gentle handling of sensitive samples.
Automatically generated - may contain errors
3 protocols using s 200 superdex column
1
Purification of PilA1 Protein Variants
2
Recombinant Nectin-4 Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HT1376, NCI-H292, PC3, and
CT26 cells were obtained from ATCC (American Type Culture Collection).
MC38 cells were obtained from the National Cancer Institute (L-159-2018/1).
CT26 and MC38 cells were engineered to express mouse Nectin-4 (NM_027893.3)
as described.21 (link) Human peripheral blood
mononuclear cell (PBMC) isolation was described.21 (link)Recombinant proteins: human CD137 (92 204B, R&D
Systems) and human CD137L (2295-4L-025, R&D Systems) were purchased.
Human Nectin-4 (residues 32–349) and rat Nectin-4 (residue
31–347) with a gp67 signal sequence and C-terminal FLAG tag
were cloned into pFastbac-1 and baculovirus using standard Bac-to-Bac
protocols (Life Technologies). Sf21 cells at 1 × 106 mL–1 in Excell-420 medium (Sigma) at 27 °C
were infected at a multiplicity of infection (MOI) of 2 with a P1
virus stock for protein expression. The supernatant was harvested
at 72 h and incubated for 1 h at 4 °C with anti-FLAG M2 affinity
agarose resin (Sigma) followed by a phosphate-buffered saline (PBS)
wash. Resin was subsequently transferred to a column and washed extensively
with phosphate-buffered saline (PBS). Protein was eluted with 100
μg/mL FLAG peptide concentrated to a volume of 2 mL and loaded
onto an S-200 Superdex column (GE Healthcare) in PBS at 1 mL/min.
2 mL fractions were collected and fractions containing Nectin-4 protein
were concentrated.
CT26 cells were obtained from ATCC (American Type Culture Collection).
MC38 cells were obtained from the National Cancer Institute (L-159-2018/1).
CT26 and MC38 cells were engineered to express mouse Nectin-4 (NM_027893.3)
as described.21 (link) Human peripheral blood
mononuclear cell (PBMC) isolation was described.21 (link)Recombinant proteins: human CD137 (92 204B, R&D
Systems) and human CD137L (2295-4L-025, R&D Systems) were purchased.
Human Nectin-4 (residues 32–349) and rat Nectin-4 (residue
31–347) with a gp67 signal sequence and C-terminal FLAG tag
were cloned into pFastbac-1 and baculovirus using standard Bac-to-Bac
protocols (Life Technologies). Sf21 cells at 1 × 106 mL–1 in Excell-420 medium (Sigma) at 27 °C
were infected at a multiplicity of infection (MOI) of 2 with a P1
virus stock for protein expression. The supernatant was harvested
at 72 h and incubated for 1 h at 4 °C with anti-FLAG M2 affinity
agarose resin (Sigma) followed by a phosphate-buffered saline (PBS)
wash. Resin was subsequently transferred to a column and washed extensively
with phosphate-buffered saline (PBS). Protein was eluted with 100
μg/mL FLAG peptide concentrated to a volume of 2 mL and loaded
onto an S-200 Superdex column (GE Healthcare) in PBS at 1 mL/min.
2 mL fractions were collected and fractions containing Nectin-4 protein
were concentrated.
3
SEC Analysis of CaM-Nanodiscs Complexes
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