Recovery from inclusion bodies

One of the main causes of artifacts in the spectroscopy of proteins is the frequent tendency of proteins to aggregate, either during the folding step in recovery from inclusion bodies or as a result of the delicate balance of solubility of many proteins. For this reason, it is important to pay strict attention to monitoring aggregation (see Strategic Planning, discussion of Clarification of Solutions). 

Methods presently employed for obtaining correctly refolded proteins from inclusion body preparations are often all-or-none propositions. They typically consist of denaturant solubilization, in urea or guanidine, followed by dilution or dialysis (2). Recovery of native activity or structure may be aided by using additives (enzyme inhibitors, co-factors, oxidation-reduction couples, etc.), which act to stabilize the native-state protein conformation. However, because such efforts are time-consuming and tedious, systematic examinations of solution conditions for protein folding/unfolding are rarely performed. 

Active Creatine Kinase Refolded from Inclusion Bodies in Escherichia coli Improved Recovery by Removal of Contaminating Protease... 

Methods. The recovery of oxidized mBST monomer from inclusion body preparations involves the dissolution of the inclusion bodies and the subsequent air oxidation and refolding of the soluble reduced protein. Protocols for the investigation of these processes are described below. All experiments were performed at 5°C to minimize mBST degradation unless otherwise noted. 

Recently, SPs were used successfully for improving the refolding of recombinant proteins expressed in bacterial hosts, in the form of inactive and insoluble aggregates known as inclusion bodies (Gautam et al, 2012). From the biotechnological perspective, the recovery of an active protein from inclusion bodies is achieved in two steps ... 

Gautam, S., Dubey, P, Singh, P, Kesavardhana, S., Varadarajan, R. and Gupta, M. N. (2012). Smart polymer mediated purification and recovery of active proteins from inclusion bodies. Journal of Chromatography A, 1235,10-25. 

Vandenbroeck et al.7 used an ELISA to determine the recovery of immu-noreactive porcine interferon-gamma (IFN-y) from E. coli inclusion bodies. The ELISA used a polyclonal coating antibody with detection by a MAb. The inclusion bodies were solubilized in diluted 6 M guanidine/HCl and IFN subsequently refolded by its removal. The antiviral activity of the interferon was measured with a bioassay using the cytopathic effect (CPE) of vesicular stomatitis virus (VSV) on bovine kidney cells. The results of this study showed that the immu-noreactivity measured by ELISA matched the biological activity measured by bioassay. 

Dempster, R.P., Robinson, C.M. and Harrison, G.B. (1996) Parasite vaccine development large-scale recovery of immunogenic Taenia ovis fusion protein GST-45W(B/X) from Escherichia coli inclusion bodies. Parasitology Research 82, 291-296. 

The basis utilized by Petrides et al.15 is 1500 kg of purified BHI per year. They indicate that this represents 10-15% of the world demand.17 In essence, the following downstream steps are involved in sequence during synthetic BHI production. The fermentation step (not a downstream step) is also included to provide some continuity. The steps are fermentation, cell harvesting, cell disruption, inclusion body recovery, inclusion body solubilization, enzymatic conversion, refolding, sulfitolysis, CNBr cleavage, final purification steps, and crystallization. In the flow chart provided by Petrides et al.15, a surge tank separates the upstream from the downstream processes. This tank is in between the fermentor and the downstream processing steps. 

Inclusion Bodies and Recovery of Proteins from the Aggregated State... 

Expression and Recovery of Soluble CK from Insoluble Inclusion Bodies. Construction of the Expression Vector, pKTCK3F. The cDNA insert encoding CK from the electric organ of Torpedo californica was isolated from... 

Conclusion. We have expressed eukaryotic CK in . coli and have demonstrated that active protein can be recovered from the resulting inclusion bodies. Based on the parameters tested, this protein appears to be very similar to CK purified from electric organ tissue. The recovery scheme is simple, involving cell lysis and overnight extraction with a detergent-containing buffer followed by sonication and isolation of... 

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