Production and Purification of Human Chemokine Receptors
Author Information
Author(s): Ren Hui, Yu Daoyong, Ge Baosheng, Cook Brian, Xu Zhinan, Zhang Shuguang
Primary Institution: Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
Hypothesis
Can human chemokine receptors CCR5, CCR3, CXCR4, and CX3CR1 be produced and purified at high levels in E. coli?
Conclusion
The study successfully achieved high-level production and purification of four human GPCR chemokine receptors in E. coli, providing sufficient quantities for structural analysis.
Supporting Evidence
- Four human chemokine receptors were successfully produced in E. coli.
- The study used a two-step assembly/amplification PCR method for gene synthesis.
- Zwitterionic detergents were found to be most effective for solubilizing the receptors.
- High-level expression was achieved using low-copy-number plasmids.
- Purification involved a two-step process using Ni2+ affinity chromatography and size exclusion chromatography.
Takeaway
The researchers figured out how to make important proteins in bacteria so they can study them better. This helps us understand diseases like HIV and cancer.
Methodology
The genes for the receptors were synthesized and expressed in E. coli using specific plasmids, followed by systematic screening of growth conditions and detergents for purification.
Limitations
The expression levels varied significantly among the different receptors, with some yielding lower amounts due to toxicity in host cells.
Digital Object Identifier (DOI)
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