Production of readily crystallizable glycoproteins in HEK293S GnTI- cell line: a case study of human natural killer cell receptors

Ondřej Vaněk1, Jan Bláha1, Barbora Kalousková1, Samuel Pažický1, Ondřej Skořepa1, Yuguang Zhao2, Karl Harlos2, Tereza Skálová3, Jan Dohnálek3

1Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 12840 Prague 2, Czech Republic

2Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, OX3 7BN Oxford, United Kingdom

3Institute of Biotechnology, Academy of Sciences of the Czech Republic, Průmyslová 595, 25242 Jesenice, Czech Republic

Recombinant protein expression can be a costly enterprise, especially for proteins that are not easily expressed in prokaryotic cells and are sometimes labelled as „difficult targets“. Here we would like to show a case study of a recombinant expression of such a difficult target – human natural killer (NK) cell receptor protein 1 and its binding partner lectin-like transcript 1, both belonging to the NK cell receptor C‑type lectin-like family.

Human embryonic kidney 293 cell line deficient in N-acetylglucosaminyltransferase I (HEK293S GnTI-) is well known tool for expression of proteins with homogeneous and deglycosylatable N‑glycosylation, a feature crucial especially for protein crystallography [1]. However, production protocol using this cell line based on transient transfection of adherent cell culture is costly to scale-up and has reportedly lower expression yields [2].

In this work we have adapted HEK293S GnTI- cell line to growth in suspension and optimized its transient transfection. While transfection at standard cultivation cell density proved very little success we have found out that concentrating the cells to high cell density substantially increases transfection efficiency, greatly enhancing protein yields and creating fast and scalable production process.

We demonstrate this on the production of soluble lectin-like transcript 1 [3] naturally present on natural killer and T‑lymphocytes, but upregulated in glioblastoma cells, one of the most lethal tumours, where it acts as a mediator of immune escape [4]. Furthermore, lectin-like transcript 1 has been recently suggested as a biomarker for B-cell non-Hodgkin’s lymphomas and connected with other cancerous manifestations [5]. The prepared soluble domain of lectin-like transcript 1 with homogeneous glycosylation was readily crystallized and following optimization of crystal conditions this protein preparation ultimately led to the first structure determination of this receptor [6].

However, both of these receptors are present on NK cells and also on circulating T and B lymphocytes, with lectin-like transcript 1 being also expressed on antigen presenting cells, suggesting a wider role of these receptors in immune regulation and response. NK cell receptor protein 1 positive lymphocytes are thus being implicated to have role in diseases connected with immune malfunctions – e.g. multiple sclerosis [7], rheumatoid arthritis [8] or Crohne’s disease [9]. In order to improve on the productivity for NK cell receptor protein 1 we are now using a stably transfected HEK293S GnTI- cell pool with a tenfold yield improvement.


This study was supported by BIOCEV (ERDF CZ.1.05/1.1.00/02.0109), Czech Science Foundation (15-15181S), Ministry of Education, Youth and Sports of the Czech Republic (LG14009), Charles University (UNCE 204025/2012, SVV 260079/2014), BioStruct-X and Instruct European infrastructure projects.


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