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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 109-117 |
| Number of pages | 9 |
| Journal | Protein Expression and Purification |
| Volume | 115 |
| DOIs | |
| State | Published - Nov 1 2015 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
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Advancing Rhodobacter sphaeroides as a platform for expression of functional membrane proteins. / Erbakan, Mustafa; Curtis, Brandon S.; Nixon, B. Tracy; Kumar, Manish; Curtis, Wayne R.
In: Protein Expression and Purification, Vol. 115, 01.11.2015, p. 109-117.Research output: Contribution to journal › Article
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.
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.
UR - http://www.scopus.com/inward/record.url?scp=84941934276&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84941934276&partnerID=8YFLogxK
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 -