This part contains the sequence coding for the RFP protein linked to two cellulose binding domains, one located at the N-terminus (CBD1 - BBa_K1934080) and one located at the C-terminus (CBD2 - BBa_K1934090). It is a RFP-CBDs generator, based on the model of our streptavidin-CBDs generator (BBa_K1934020).
The part BBa_K1934000 was designed for two reasons:
- RFP properties and especially its red coloration enables the follow the purification process visually;
- this colorimetric signal allows to determine if the covalent linkage of the CBDs on the protein disrupts the RFP folding.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 380
Illegal AgeI site found at 955
Illegal AgeI site found at 1067
- 1000COMPATIBLE WITH RFC
Purification Using Cellulose Affinity
The BBa_K1934000 part, conceived by the 2016 INSA-Lyon team and synthesized by IDT, was cloned into pSB1C3 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mmol.L-1 and glucose 5 mmol.L-1. Cells were harvested and resuspended in 1 mL lysis buffer (50 mmol.L-1 Tris, 300 mmol.L-1 NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14,000 g for 10 min. The supernatant was treated as follow:
- Wash microcrystalline cellulose five time in water. Then equilibrate in wash buffer (ammonium sulfate 1 mol.L-1). Pack the cellulose (10x10 mm) in small chromatography columns (we used syringes barrels).
- Gently pour the lysate supernatant on the column. Once the liquid starts flowing out evenly, measure the OD280 of the different fractions. Continue pouring wash buffer until the OD280 stabilizes around zero.
- Change the washing buffer to water. OD280 shortly rises. Keep the fractions with highest OD280. They should contain the protein.
- Analyze collected fractions on a SDS-PAGE. ,/ul> Optionally, proteins may be concentrated using ultrafiltration.
Verification of protein foldingThe RFP-CBDs was efficiently purified thanks to a cellulose column. A fluorescence signal is measured proving that the RFP is still functional even with the CBDs covalent ligation.
ConclusionThis experiment shows that the presence of CBDs on RFP-CBDs does not impair neither the cellulose binding nor the protein folding.