title: Advancing Rhodobacter sphaeroides as a platform for expression of functional membrane proteins.
abstract: Membrane protein overexpression is often hindered by toxic effects on the expression host, limiting achievable volumetric productivity. Moreover, protein structure and function may be impaired due to inclusion body formation and proteolytic degradation. To address these challenges, we employed the photosynthetic bacterium, Rhodobacter sphaeroides for expression of challenging membrane proteins including human aquaporin 9 (hAQP9), human tight junction protein occludin (Occ), Escherichia coli toxin peptide GhoT, cellulose synthase enzyme complex (BcsAB) of R. sphaeroides and cytochrome-cy (Cyt-cy) from Rhodobacter capsulatus. Titers of 47 mg/L for Cyt-cy, 7.5 mg/L for Occ, 1.5 mg/L for BcsAB and 0.5 mg/L for hAQP9 were achieved from affinity purification. While purification of GhoT was not successful, transformants displayed a distinct growth phenotype that correlated with GhoT expression. We also evaluated the functionality of these proteins by performing water transport studies for hAQP9, peroxidase activity for cytochrome-cy, and in vitro cellulose synthesis activity assay for BcsAB. While previous studies with Rhodobacter have utilized oxygen-limited semi-aerobic growth for membrane protein expression, substantial titer improvements are achieved as a result of a 3-fold increase in biomass yield using the anaerobic photoheterotrophic growth regime, which utilizes the strong native puc promoter. This versatile platform is shown to enable recovery of a wide variety of difficult-to-express membrane proteins in functional form.
authors.affiliation: Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address: mxe198@psu.edu.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Erbakan
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California at Berkeley, Berkeley, CA 94720-1462, United States. Electronic address: brandon.curtis@berkeley.edu.
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Curtis
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address: btn1@psu.edu.
authors.forename: B Tracy
authors.initials: BT
authors.lastname: Nixon
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address: manish.kumar@psu.edu.
authors.forename: Manish
authors.initials: M
authors.lastname: Kumar
authors.affiliation: Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802, United States; Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, United States. Electronic address: wrc2@psu.edu.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Curtis
mesh_terms: D001709:Biotechnology / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins
keywords: Anaerobic photoheterotrophic growth; Aquaporin; Cellulose synthase; Cytochrome; Membrane protein; Rhodobacter sphaeroides; Toxin–antitoxin