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High-level expression and purification of soluble form of human natural killer cell receptor NKR-P1 in HEK293S GnTI(-) cells
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SYSNO ASEP 0489434 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title High-level expression and purification of soluble form of human natural killer cell receptor NKR-P1 in HEK293S GnTI(-) cells Author(s) Bláha, J. (CZ)
Kalousková, B. (CZ)
Skořepa, O. (CZ)
Pažický, S. (CZ)
Novák, Petr (MBU-M) RID, ORCID
Vaněk, O. (CZ)Source Title Protein Expression and Purification. - : Elsevier - ISSN 1046-5928
Roč. 140, DEC 2017 (2017), s. 36-43Number of pages 8 s. Language eng - English Country US - United States Keywords NKR-P1 ; CD161 ; klrb1 Subject RIV CE - Biochemistry OECD category Biochemistry and molecular biology Institutional support MBU-M - RVO:61388971 UT WOS 000411303200005 EID SCOPUS 85026659195 DOI 10.1016/j.pep.2017.07.016 Annotation Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor, and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFN gamma. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI(-) cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2019
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