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.

Acetylesterase CE16 was identified as a part of the enzymatic cocktail secreted by fungus Hypocrea jecorina (anamorph: Trichoderma reesei) during its growth on cellulose. Later it was classified as the first member of a newly organized carbohydrate esterase family CE16. Further studies showed that acetylesterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. To study the relationship between structure and function of acetylesterase, highly purified recombinant enzyme produced by Trichoderma reesei Rut C-30 was prepared. The enzyme was composed of 348 amino acid residues from which the 1 -19 formed a secretion signal peptide. Determined molecular mass of purified recombinant acetylesterase (Aes1) was 45 kDa which was more than molecular mass calculated from amino acid sequence. As it has been proved later, the difference was caused by the enzyme glycosylation. Glycosylation of proteins increases their stability, but it can also be a source of heterogeneity, which might be a problem during crystallization. To make the future X-ray study of the enzyme easier, recombinant non-glycosylated enzyme needed to be prepared. For these purposes, a synthetic gene optimized for protein expression in Escherichia coli was designed and synthetized. The first nonglycosylated acetylesterase obtained by the expression of its synthetic gene in E. coli cells was mostly insoluble or aggregated. Conditions of cell cultivation, induction of gene expression and cells disruption were necessary to optimize. Presently, after optimization of all mentioned steps, the non-glycosylated recombinant CE16 acetylesterase was prepared in the soluble and active form, ready for further downstream procedures, involving protein purification and crystallization.