A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.
Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent dru...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3149604?pdf=render |
| _version_ | 1828305333753217024 |
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| author | Jiangping Bai Douglas J Swartz Irina I Protasevich Christie G Brouillette Patina M Harrell Ellen Hildebrandt Brigitte Gasser Diethard Mattanovich Andrew Ward Geoffrey Chang Ina L Urbatsch |
| author_facet | Jiangping Bai Douglas J Swartz Irina I Protasevich Christie G Brouillette Patina M Harrell Ellen Hildebrandt Brigitte Gasser Diethard Mattanovich Andrew Ward Geoffrey Chang Ina L Urbatsch |
| author_sort | Jiangping Bai |
| collection | DOAJ |
| description | Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.Codon-optimized "Opti-Pgp" and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T(m) ∼40 °C to 49 °C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins. |
| first_indexed | 2024-04-13T14:26:50Z |
| format | Article |
| id | doaj.art-f424fe6f9d1a45adb472c97182b63619 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T14:26:50Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-f424fe6f9d1a45adb472c97182b636192022-12-22T02:43:18ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0168e2257710.1371/journal.pone.0022577A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.Jiangping BaiDouglas J SwartzIrina I ProtasevichChristie G BrouillettePatina M HarrellEllen HildebrandtBrigitte GasserDiethard MattanovichAndrew WardGeoffrey ChangIna L UrbatschStructural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.Codon-optimized "Opti-Pgp" and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T(m) ∼40 °C to 49 °C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins.http://europepmc.org/articles/PMC3149604?pdf=render |
| spellingShingle | Jiangping Bai Douglas J Swartz Irina I Protasevich Christie G Brouillette Patina M Harrell Ellen Hildebrandt Brigitte Gasser Diethard Mattanovich Andrew Ward Geoffrey Chang Ina L Urbatsch A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. PLoS ONE |
| title | A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. |
| title_full | A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. |
| title_fullStr | A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. |
| title_full_unstemmed | A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. |
| title_short | A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. |
| title_sort | gene optimization strategy that enhances production of fully functional p glycoprotein in pichia pastoris |
| url | http://europepmc.org/articles/PMC3149604?pdf=render |
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