Heterologous expression and purification of the bicarbonate transporter BicA from Synechocystis sp. PCC 6803

The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and p...

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Bibliographic Details
Published in:Protein expression and purification 2020-11, Vol.175, p.105716-105716, Article 105716
Main Authors: Bu, Guanhong, Parrish, Sydney, Gleason, Patrick R., Nielsen, David R., Nannenga, Brent L.
Format: Article
Language:English
Subjects:
Bacterial Proteins - biosynthesis
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Bicarbonate transporter
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Expression
Membrane protein
Protein overexpression
Protein purification
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Sodium-Bicarbonate Symporters - biosynthesis
Sodium-Bicarbonate Symporters - chemistry
Sodium-Bicarbonate Symporters - genetics
Sodium-Bicarbonate Symporters - isolation & purification
Synechocystis - genetics
Synechocystis - metabolism
Synechocystis sp.PCC 6803
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Summary:The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA6803) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA6803 relative to other strains screened, and membrane solubilization with n-dodecyl-β-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA6803. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture. •Expression and purification of BicA from Synechocystis sp. PCC 6803 was optimized.•The use of C43 (DE3) Rosetta2 cells produced the highest levels of BicA6803.•The detergent DDM was found to be the most suitable detergent tested for the purification of BicA6803.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2020.105716