Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

• Klíčová slova
• Thermofluor assay, crystallizability optimization, crystallization, differential scanning fluorimetry, oligomerization, single-chain antibody fragment,
• MeSH
• gelová chromatografie MeSH
• jednořetězcové protilátky chemie   MeSH
• krystalizace * MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jednořetězcové protilátky MeSH

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.

• MeSH
• bioreaktory * MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• enzymy metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• imunoglobulin G metabolismus   MeSH
• imunoglobuliny - fragmenty chemie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• enzymy MeSH
• imunoglobulin G MeSH
• imunoglobuliny - fragmenty MeSH

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 IFNγ. 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.

• Klíčová slova
• CD161, HEK293, LLT1, NK cells, NKR-P1, klrb1,
• MeSH
• bioreaktory MeSH
• buňky NK metabolismus   MeSH
• buňky Th17 metabolismus   MeSH
• Escherichia coli genetika   MeSH
• HEK293 buňky MeSH
• lektinové receptory NK-buněk - podrodina B biosyntéza   genetika   izolace a purifikace   MeSH
• lektiny typu C metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• receptory buněčného povrchu metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CLEC2D protein, human MeSH Prohlížeč
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• receptory buněčného povrchu MeSH

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.

• Klíčová slova
• DhmeA, Haloferax mediterranei, catalysis, cryo-EM, haloalkane dehalogenase, multimerization, x-ray crystallography,
• MeSH
• elektronová kryomikroskopie metody   MeSH
• hydrolasy * chemie   MeSH
• krystalografie rentgenová MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy * MeSH