High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1
Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most...
| Main Authors: | , , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
Public Library of Science
2010
|
| Online Access: | http://hdl.handle.net/1721.1/52391 |
| _version_ | 1811082558121705472 |
|---|---|
| author | Xu, Zhinan Cook, Brian L. Ge, Baosheng Yu, Daoyong Ren, Hui Zhang, Shuguang |
| author2 | Massachusetts Institute of Technology. Center for Biomedical Engineering |
| author_facet | Massachusetts Institute of Technology. Center for Biomedical Engineering Xu, Zhinan Cook, Brian L. Ge, Baosheng Yu, Daoyong Ren, Hui Zhang, Shuguang |
| author_sort | Xu, Zhinan |
| collection | MIT |
| description | Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis. |
| first_indexed | 2024-09-23T12:05:19Z |
| format | Article |
| id | mit-1721.1/52391 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T12:05:19Z |
| publishDate | 2010 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/523912022-09-28T00:01:41Z High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 Xu, Zhinan Cook, Brian L. Ge, Baosheng Yu, Daoyong Ren, Hui Zhang, Shuguang Massachusetts Institute of Technology. Center for Biomedical Engineering Zhang, Shuguang Xu, Zhinan Cook, Brian L. Ge, Baosheng Yu, Daoyong Ren, Hui Zhang, Shuguang Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis. 2010-03-08T19:40:00Z 2010-03-08T19:40:00Z 2009-02 2008-10 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/52391 Ren, Hui et al. “High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1.” PLoS ONE 4.2 (2009): e4509. en_US http://dx.doi.org/10.1371/journal.pone.0004509 PLoS ONE Creative Commons Attribution http://creativecommons.org/licenses/by/2.5/ application/pdf Public Library of Science PLoS |
| spellingShingle | Xu, Zhinan Cook, Brian L. Ge, Baosheng Yu, Daoyong Ren, Hui Zhang, Shuguang High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 |
| title | High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 |
| title_full | High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 |
| title_fullStr | High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 |
| title_full_unstemmed | High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 |
| title_short | High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1 |
| title_sort | high level production solubilization and purification of synthetic human gpcr chemokine receptors ccr5 ccr3 cxcr4 and cx3cr1 |
| url | http://hdl.handle.net/1721.1/52391 |
| work_keys_str_mv | AT xuzhinan highlevelproductionsolubilizationandpurificationofsynthetichumangpcrchemokinereceptorsccr5ccr3cxcr4andcx3cr1 AT cookbrianl highlevelproductionsolubilizationandpurificationofsynthetichumangpcrchemokinereceptorsccr5ccr3cxcr4andcx3cr1 AT gebaosheng highlevelproductionsolubilizationandpurificationofsynthetichumangpcrchemokinereceptorsccr5ccr3cxcr4andcx3cr1 AT yudaoyong highlevelproductionsolubilizationandpurificationofsynthetichumangpcrchemokinereceptorsccr5ccr3cxcr4andcx3cr1 AT renhui highlevelproductionsolubilizationandpurificationofsynthetichumangpcrchemokinereceptorsccr5ccr3cxcr4andcx3cr1 AT zhangshuguang highlevelproductionsolubilizationandpurificationofsynthetichumangpcrchemokinereceptorsccr5ccr3cxcr4andcx3cr1 |