Research Article His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss? Elena Krachmarova, 1,2 Milena Tileva, 1,2 Elena Lilkova, 2,3 Peicho Petkov, 2,4 Klaus Maskos, 5 Nevena Ilieva, 2,3 Ivan Ivanov, 1,2 Leandar Litov, 2,4 and Genoveva Nacheva 1,2 1 Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, 1113 Sofa, Bulgaria 2 Institute for Interdisciplinary Research and Technologies, 1421 Sofa, Bulgaria 3 Institute of Information and Communication Technologies, Bulgarian Academy of Sciences, 1113 Sofa, Bulgaria 4 Faculty of Physics, Sofa University “St. Kliment Ohridski”, 1164 Sofa, Bulgaria 5 Proteros Biostructures, 82152 Martinsried, Germany Correspondence should be addressed to Genoveva Nacheva; genoveva@bio21.bas.bg Received 1 March 2017; Accepted 23 April 2017; Published 8 June 2017 Academic Editor: Rituraj Purohit Copyright © 2017 Elena Krachmarova et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In order to obtain glycosylated human interferon-gamma (hIFN) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purifcation, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His 6 -FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in E. coli cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. Te latter showed that the tags interact with the receptor binding domains and the fexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFN receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site. 1. Introduction Te choice of optimal expression conditions and protocol for purifcation of recombinant proteins depends on the characteristics of the protein of interest, the experimental needs, and the further application of the purifed product. Many proteins need to be modifed following translation in order to become active. A large number of recombinant proteins with clinical relevance are naturally glycosylated [1]. Tis posttranslational modifcation plays important role in protein folding, stability, and protease resistance in the blood stream and is exclusively carried out by eukaryotic cells. Many eukaryotic hosts such as mammalian, insect, yeast, and plant cells have been developed to express glycosylated proteins, and each has their relative advantages and disadvantages [2]. Although the early steps of glycosylation in the endo- plasmic reticulum (ER) are very similar across eukaryotes, the structure of the fnal glycans is species specifc due to diferences in the oligosaccharide processing pathways [2, 3]. Expression of glycoproteins in mammalian cells will result in mammalian-type glycosylation. For human proteins this is ideal; however some cell lines will add the nonhuman Gal 1-3 Gal epitope and the -glycolylneuraminic acid. Insect expression systems will add shorter -glycans, with little sialylation. Plant cells typically produce glycans that contain extra fucose and xylose residues [4]. Yeast expression systems have a very diferent glycosylation pattern from mammalian cells, with only mannose-containing glycans [5]. Human interferon-gamma (hIFN) is a secretory gly- coprotein, which plays key role in the regulation of innate and adaptive immunity. It is composed of 143 amino acid residues (17 kDa) and is a rare case of cysteinless protein. Te biologically active form of hIFN is a homodimer where the two monomers are bound in antiparallel direction. Te Hindawi BioMed Research International Volume 2017, Article ID 3018608, 12 pages https://doi.org/10.1155/2017/3018608