Journal of Chromatography A, 1235 (2012) 10–25 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Smart polymer mediated purification and recovery of active proteins from inclusion bodies Saurabh Gautam a , Priyanka Dubey a , Pranveer Singh b , S. Kesavardhana b , Raghavan Varadarajan b , Munishwar N. Gupta a,∗ a Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India b Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India a r t i c l e i n f o Article history: Received 6 December 2011 Received in revised form 16 February 2012 Accepted 21 February 2012 Available online 3 March 2012 Keywords: Affinity precipitation CcdB MBP Protein refolding Pseudochaperonin Smart polymer a b s t r a c t Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacte- rial 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 340 nm (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 8 mg L -1 (for CD4D12, the first two domains of human CD4) to 58 mg L -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 pseu- dochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50 min 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 emis- sion spectra, circular dichroism (CD) spectroscopy, melting temperature (T m ), 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. © 2012 Elsevier B.V. All rights reserved. 1. Introduction While it is well established that a correctly folded conforma- tion of a protein – called the native structure – is responsible for its biological activity [1], the exact mechanisms are still less than com- pletely understood. The well known work by Anfinsen showed that the information for folding resides in the primary sequence of the protein [2]. As Hartl et al. [3] recently observed “Although small pro- teins may fold at very fast speeds (within microseconds), in dilute buffer solutions, larger multidomain proteins may take minutes to hours to fold, and often even fail to reach their native states in vitro”. In vivo, protein crowding [4] contributes to aggregation of non- native structures. This is prevented by molecular chaperones or chaperonins in a cell. Their role is not always limited to prevention of aggregation, but may extend to acceleration of folding and rever- sal of misfolding events [3]. Many excellent reviews are available on ∗ Corresponding author. Tel.: +91 11 2659 1503; fax: +91 11 2658 1073. E-mail address: appliedbiocat@yahoo.co.in (M.N. Gupta). protein folding [5,6]. While Sinha and Udgaonkar [5] have provided a rigorous treatment of early events in protein folding, Nickson and Clarke [6] have reviewed both theoretical and experimental meth- ods (and their results) used to study protein folding. There is enough evidence that protein folding involves existence of one or more partially folded structures. In many cases, it is possible to isolate ‘molten globules’ which occur on the folding pathway. The ‘oil drop’ model of protein structure envisages that there is a hydrophobic core with polar amino acids on the surface H-bonded with water. Hydrophobic clusters do occur on the protein surface and are quite often part of a specific binding site for ligands/substrates. Apart from the above ‘nucleation model’, ‘energy landscape model’ has also been proposed more recently, where folding intermediates are viewed as ‘kinetic traps’ on the folding pathway. The greater under- standing of protein folding is also of practical utility in the context of protein refolding. The overexpression of recombinant proteins in bacterial hosts often leads to the formation of inactive and insolu- ble aggregates called inclusion bodies. In some cases, proteins in these inclusion bodies may not be completely inactive [7]. Pro- tein aggregation as such has also attracted attention as the cause behind several neurodegenerative diseases and cataract formation 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.02.048