TY - JOUR
T1 - Advancing Rhodobacter sphaeroides as a platform for expression of functional membrane proteins
AU - Erbakan, Mustafa
AU - Curtis, Brandon S.
AU - Nixon, B. Tracy
AU - Kumar, Manish
AU - Curtis, Wayne R.
N1 - Funding Information:
We thank Jeff Larsen for his efforts in molecular biology training. Deborah Hansen and Philip Laible (Argonne National Labs. University of Chicago, Chicago, IL) kindly provided Rhodobacter sphaeroides strains (ATCC17023 and PUC705-BA) and the expression vector (pRKPLHT7). We also acknowledge Tucker Langseth and Cory Thomas for culturing assistance; Hsin-Yao Cheng and Professor Thomas Wood (Penn State) for providing GhoT and GhoTF38R genes; Juan Du for assisting with BcsAB activity assay; Penn State Institutes of Energy and Environment for equipment support of TEM and stopped flow. This project was initiated with funding from the Penn State’s Grace Woodward Grants for Collaborative Research and Medicine, which included providing the occludin codon-optimized gene construct. A fellowship to B.R.C. from the Department of Chemical Engineering BioEndowment fund provided full time summer research support at this early stage of assessment. An Advanced Research Project Agency (ARPA-Electrofuels, #DE-AR0000092) grant “Development of Rhodobacter as a fuels production platform” to W.R.C. provided support for genetic transformation development work.
Funding Information:
M.E. was supported for the majority of this work by a grant from the Turkish Republic, Ministry of National Education . The Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy , Office of Science, and Office of Basic Energy Sciences under Award Number DE-SC0001090 provided follow up support to M.E. for characterization of Rhodobacter cellulose synthase.
Publisher Copyright:
© 2015 Published by Elsevier Inc.
PY - 2015/11/1
Y1 - 2015/11/1
N2 - 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.
AB - 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.
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U2 - 10.1016/j.pep.2015.05.012
DO - 10.1016/j.pep.2015.05.012
M3 - Article
C2 - 26008117
AN - SCOPUS:84941934276
VL - 115
SP - 109
EP - 117
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -