Improvement of downstream processing of proteins by means of genetic engineering methods

Universität Bielefeld, Technische Fakultät, Arbeitsgruppe Fermentationstechnik, Postfach 10 01 31, 4800 Bielefeld 1, Germany
Biotechnology Advances (Impact Factor: 9.02). 02/1993; 11(1):31-77. DOI: 10.1016/0734-9750(93)90409-G
Source: PubMed


The rapid advancement of genetic engineering has allowed to produce an impressive number of proteins on a scale which would not have been achieved by classical biotechnology. At the beginning of this development research was focussed on elucidating the mechanisms of protein overexpression. The appearance of inclusion bodies may illustrate the success. In the meantime, genetic engineering is not only expected to achieve overexpression, but to improve the whole process of protein production. For downstream processing of recombinant proteins, the synthesis of fusion proteins is of primary importance. Fusion with certain proteins or peptides may protect the target protein from proteolytic degradation and may alter its solubility. Intracellular proteins may be translocated by means of fusions with signal peptides. Affinity tags as fusion complements may render protein separation and purification highly selective. These methods as well as similar ones for improving the downstream processing of proteins will be discussed on the basis of recent literature.

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Available from: Karl Friehs, Oct 03, 2015
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    • "Expression as inclusion bodies has some beneficial effect in some specific circumstances. Formations of IB can facilitate downstream processes such as purification of recombinant proteins (Flaschel, 1993). In the case of formation of IBs, in vitro refolding is a selective strategy for converting the inactive and insoluble inclusion bodies into soluble form, correctly folded and biologically active products. "
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    ABSTRACT: Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process. In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA) in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and Dithiothreitol (DTT) on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, L-arginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM. Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presence of higher concentration of L-arginine (> 500 mM) or pH (> 10) is not associated with higher activity.
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