From inclusion bodies

FIG. 20-87 Illustration of a refolding process for a protein from inclusion body. 

Titchener-Hooker, N. J., Gritsis, D., Mannweiler, K., Olbrich, R., Gardiner, S. A. M., Fish, N. M., and Hoare, M., Integrated process design for producing and recovering proteins from inclusion bodies, BioPharm, July/Aug., 34,1991. 

Muller, C. and Rinas, U., Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography, /. Chromatogr. A, 855, 203, 1999. 

Besides fhe application in chromatography, chaotropic substances are used for protein extraction, e.g., of recombinant proteins from inclusion bodies and for precipitation by salts or organic solvents... 

Wang, P.-R Novak, W.R.P. Cantwell, J.S. Babbitt, P.C. McLeish, M.J. Kenyon, G.L. Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies. Protein Expr. Purif., 26, 89-95 (2002)... 

S.J. Ellington, W.R. Chapman, M.S. Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase a model for studying phosphagen kinases. Protein Sci., 6, 444-449 (1997)... 

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). 

A major class of insoluble proteins are recombinant proteins expressed (usually in Escherichia coli) as inclusion bodies. These are dense aggregates found inside cells that consist mainly of a desired recombinant product, but in a nonnative state. Inclusion bodies may form for a variety of reasons, such as insolubility of the product at the concentrations being produced, inability to fold correctly in the bacterial environment, or inability to form correct, or any, disulfide bonds in the reducing intracellular environment. Their purification is simple, since the inclusion bodies can be separated by differential centrifugation from other cellular constituents, giving almost pure product the problem is that the protein is not in a native state, and is insoluble. Some methods for obtaining an active product from inclusion bodies are described in Coligan et al. (2001). 

The events leading to the macromolecular associations observed with (3-lactoglobulin appear to be mediated by disulfide interactions. The initial solution proposed to reduce thermoinduced aggregation was to remove the Cys 121 and replace it with alanine (Cho et al., 1994). Unfortunately, recombinant protein could not be purified from inclusion bodies. The alternative... 

Fig. 4. Visualization of several purified RANTES proteins on a Coomassie stained SDS page Lanes 1 and 2, 6xHis-tagged RANTES expressed in the periplasmic space of E. coli Lane 3, Met-RANTES expressed in the cytoplasm of E. coli and purified from inclusion bodies Lane 4, eucaryotic expressed 6xHis-tagged RANTES.

This was accomplished by reducing and carboxymethylating the free sulfhydryl groups in the proteins from inclusion bodies and subsequently purifying the desired somatotropin away from the E. coli proteins by RP-HPLC. These results yielded 0.22 and 0.27 moles of e-N-acetyllysine per mole of rpST and rbST, respectively. These data showed that approximately 25 % of the somatotropin present in the inclusion bodies contained this modified amino acid. 

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. 

Sites within the carboxy-terminal domain core of phage T4 lysozyme were substituted singly and as a group with methionine to produce a simplified core sequence. The properties of such mutant lysozymes are briefly described. In addition we describe a method to isolate mutant protein from inclusion bodies and a sensitive enzymatic assay to detect small differences in mutant protein activities. 

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

Recombinant proteins expressed at high levels in bacterial hosts are often found in the form of inclusion bodies (f). These inclusion bodies consist of dense masses of partially folded, reduced protein. In this state, the target proteins are inactive the inclusion bodies must be dissolved and the soluble protein must be refolded and oxidized into the native, active state. The typical downstream process for recovering protein from inclusion bodies includes two distinct operations the dissolution of the inclusion bodies at high concentrations of denaturant such as urea or guanidine hydrochloride followed by a dilution or gradual removal of the denaturant to permit folding and oxidation to occur ( - ). 

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. 

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