How to approach heterogeneous protein expression for biotechnological use: An overview

Abstract

Production of recombinant proteins in Escherichia coli expression systems has shown many advantages, as well as disadvantages, especially for biotechnological and other down-stream applications. The choice of an appropriate vector depends on the gene, to be cloned for purification procedures and other analyses. Selection of a suitable production strain plays an important role in the preparation of recombinant proteins. The main criteria for the selection of the host organism are the properties of the recombinant produced protein, its subsequent use and the total amount desired. The most common problems in eukaryotic gene expression and recombinant proteins purification are, for instance, post-translational modifications, formation of disulphide bonds, or inclusion bodies. Obtaining a purified protein is a key step enabling further characterization of its role in the biological system. Moreover, methods of protein purification have been developed in parallel with the discovery of proteins and the need for their studies and applications. After protein purification, and also between the individual purification steps, it is necessary to test protein stability under different conditions over time. Shortly, all the essential points have been briefly discussed, which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source and expressed heterogeneously in prokaryotic production system.

Two Simple Methods for Optimizing the Production of "Difficult-to-Express" GnRH-DFF40 Chimeric Protein

“…16 The presence of disulfide bonds in DFF40 molecule and its prone to aggregation identity or formation of inclusion body is a key problem. 6,17 Besides, reductive condition of the bacterial cytoplasm and the DNase nature of DFF40 that cause cytotoxicity to bacterial host cells are other challenges. 18 One approach to overcome these limitations is selection of an optimal medium that helps to obtain a high cell density of bacterial population and retains plasmid stability at a high rate.…”

“…Because synthetic form has a higher similarity with the genetic structure of E. coli and therefore higher compatibility for inheritance to bacterial progenies during the growth phase. 17…”

Guidelines to reach high-quality purified recombinant proteins

“…The first one is to avoid storage of the protein at pH values close to its isoelectric point (Table 1); otherwise, protein will precipitate. Drastic changes in pH and temperature, and overly high concentrations, should also be avoided as they may promote protein denaturation/precipitation (Jamrichová et al 2017). Buffers reported as a source of sample heterogeneity, such as Tris, should not be used (Boivin et al 2013).…”

“…In this last case, samples must be filter-sterilized (through a 0.22-μm filter with low binding capacity), or supplemented with antibacterial and antimycotic agents (e.g. 0.1% sodium azide), to avoid microbial contamination (Jamrichová et al 2017). The most suitable storage temperature can be determined experimentally by monitoring the stability of small aliquots of the protein over time at some relevant temperatures (i.e.…”

“…1 mM DTT), protein-specific ligands or serum albumins (e.g. BSA, 10 mg/ml) (Jamrichová et al 2017). At the end, the optimal storage conditions can differ significantly from the application conditions, and thus, a buffer exchange step may be necessary, followed by verification of the homogeneity, functionality and concentration of the protein.…”