Optimized gene synthesis, high level expression, isotopic enrichment, and refolding of human interleukin-5.
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.
Mehta
D V
DV
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, USA.
DiGate
R J
RJ
Banville
D L
DL
Guiles
R D
RD
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D006801:Humans; D015848:Interleukin-5 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D014764:Viral Proteins
D015848:Interleukin-5; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases