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

Article · February 1993with151 Reads
DOI: 10.1016/0734-9750(93)90409-G · Source: PubMed
Abstract
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.
    • The main intention is to facilitate downstream purification; however gene fusions may also improve solubility and proteolytic stability and assist in refolding (Waugh 2005). There are many fusion partners for which commercially available purification systems exist, ranging in size from a few amino acids to whole proteins (Flaschel & Friehs 1993; Terpe 2003). A commonly used purification handle is the poly-histidine (His) tag, enabling purification of the recombinant protein on a column with immobilized metal ions (Hochuli et al. 1988).
    Full-text · Chapter · Jan 2012 · Biotechnology and Bioengineering
    • 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.
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Oct 2011
    • Proteolytic degradation of biopharmaceuticals before their recovery from the host-cell prevents their use as therapeutics (Murby et al., 1996 ). Locating cleavage sites within oligopeptides could enable improved stability via protein engineering (Flaschel and Friehs, 1993). To date, the PeptideCutter algorithm at ExPASy (http://www.expasy.ch) is the only tool available publicly for use in prediction of Factor Xa cleavage sites in proteins.
    [Show abstract] [Hide abstract] ABSTRACT: This paper presents an algorithm which is able to extract discriminant rules from oligopeptides for protease proteolytic cleavage activity prediction. The algorithm is developed using genetic programming. Three important components in the algorithm are a min-max scoring function, the reverse Polish notation (RPN) and the use of minimum description length. The min-max scoring function is developed using amino acid similarity matrices for measuring the similarity between an oligopeptide and a rule, which is a complex algebraic equation of amino acids rather than a simple pattern sequence. The Fisher ratio is then calculated on the scoring values using the class label associated with the oligopeptides. The discriminant ability of each rule can therefore be evaluated. The use of RPN makes the evolutionary operations simpler and therefore reduces the computational cost. To prevent overfitting, the concept of minimum description length is used to penalize over-complicated rules. A fitness function is therefore composed of the Fisher ratio and the use of minimum description length for an efficient evolutionary process. In the application to four protease datasets (Trypsin, Factor Xa, Hepatitis C Virus and HIV protease cleavage site prediction), our algorithm is superior to C5, a conventional method for deriving decision trees.
    Article · Dec 2003
  • Article · Biotechnology and Bioengineering
  • [Show abstract] [Hide abstract] ABSTRACT: The principle of metal chelate-protein interaction was applied for protein extraction by means of reverse micellar phases. For this purpose, an affinity surfactant was synthesized exposing iminodiacetic acid as the hydrophilic moiety. The application of this substance as a cosurfactant leads to enhanced extraction of proteins exhibiting histidine groups on their surface.
    Article · May 1994
  • [Show abstract] [Hide abstract] ABSTRACT: The mechanism of extraction of rat cytochrome b(5) from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b(5) and modified proteins with substitutions of the type glutamic acid --> lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. (c) 1994 John Wiley & Sons, Inc.
    Article · Sep 1994
    Paulo J Martel+1 more author...
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