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Smart polymer mediated purification and recovery of active proteins from inclusion bodies

Gautam, Saurabh, Dubey, Priyanka, Singh, Pranveer, Kesavardhana, S., Varadarajan, Raghavan, Gupta, Munishwar N.
Journal of chromatography 2012 v.1235 pp. 10-25
binding proteins, bioactive properties, chromatography, disulfide bonds, fluorescence, hosts, humans, hydrophobicity, inclusion bodies, light scattering, maltose, melting point, molecular weight, polymers, protein synthesis, recombinant proteins, signal peptide, solubilization, spectroscopy
Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8mgL⁻¹ (for CD4D12, the first two domains of human CD4) to 58mgL⁻¹ (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseudochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (Tₘ), and surface hydrophobicity measurement by ANS (8-anilino1-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding.