Optimized Gene Synthesis, High Level Expression, Isotopic Enrichment, and Refolding of Human Interleukin-5
| Autor: | Deepa V. Mehta, Debra L. Banville, R. D. Guiles, Russell J. DiGate |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Isopropyl Thiogalactoside
Protein Denaturation Protein Folding Magnetic Resonance Spectroscopy Protein Conformation Molecular Sequence Data Gene Expression Biology Inclusion bodies Viral Proteins chemistry.chemical_compound Protein structure Gene expression Genes Synthetic medicine Humans T7 RNA polymerase Cloning Molecular Base Sequence Oligonucleotide DNA DNA-Directed RNA Polymerases Nuclear magnetic resonance spectroscopy chemistry Biochemistry Protein Biosynthesis Protein folding Interleukin-5 Plasmids Biotechnology medicine.drug |
| Zdroj: | Protein Expression and Purification. 11:86-94 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.1997.0785 |
| Popis: | Structural studies on soluble proteins using nuclear magnetic resonance (NMR) spectroscopy and other structural methods in general require large quantities of isotopically enriched proteins. Human interleukin-5 is a disulfide-linked homodimeric cytokine implicated in asthmatic response. The development of a high yield overexpression system for human interleukin-5 is an important prerequisite to using modern multidimensional NMR in the characterization of the solution structure of the protein and to characterize interactions with a soluble receptor domain. Significant amounts of the protein were expressed using an optimized synthetic gene in a high yield expression system. Gene synthesis was accomplished through the ligation of six oligonucleotides composed of optimized codons. The ligated fragments were further amplified by a polymerase chain reaction and then subcloned into the T7 RNA polymerase based overexpression vector pET11a. However, the induced protein accumulated in the form of inclusion bodies. Initially, the protein was solubilized under denaturing conditions and purified in these denaturing conditions by passage through a single S-200 HR sizing column. Finally, protein refolding was initiated in the presence of 2 M urea followed by dialysis. This protocol yielded 40 mg of biologically active, isotope-enriched protein from 4 liters of minimal medium thus facilitating structural studies by NMR. The strategy described may be of immense value in the production of significant quantities of recombinant, eukaryotic proteins for structural and other studies. |
| Databáze: | OpenAIRE |
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