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

Guanhong Bu; Sydney Parrish; Patrick R Gleason; David R Nielsen; Brent L Nannenga

doi:10.1016/j.pep.2020.105716

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

Protein Expr Purif. 2020 Nov:175:105716. doi: 10.1016/j.pep.2020.105716. Epub 2020 Jul 29.

Authors

Guanhong Bu¹, Sydney Parrish², Patrick R Gleason³, David R Nielsen², Brent L Nannenga⁴

Affiliations

¹ Chemical Engineering, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85287, USA; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA.
² Chemical Engineering, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85287, USA.
³ School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287, USA; Center for Molecular Design and Biomimetics, The Biodesign Institute, Arizona State University, Tempe, AZ, 85278, USA.
⁴ Chemical Engineering, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85287, USA; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA. Electronic address: [email protected].

PMID: 32738437
DOI: 10.1016/j.pep.2020.105716

Abstract

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.

Keywords: Bicarbonate transporter; Escherichia coli; Membrane protein; Protein overexpression; Protein purification; Synechocystis sp.PCC 6803.

MeSH terms

Bacterial Proteins* / biosynthesis
Bacterial Proteins* / chemistry
Bacterial Proteins* / genetics
Bacterial Proteins* / isolation & purification
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression*
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

Substances

Bacterial Proteins
Recombinant Proteins
Sodium-Bicarbonate Symporters