BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.

• Klíčová slova
• Protein functionality, Protein quality control, Purification strategies, Recombinant proteins,
• MeSH
• chromatografie afinitní MeSH
• rekombinantní proteiny chemie   genetika   MeSH
• reprodukovatelnost výsledků * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• rekombinantní proteiny MeSH

The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.

• Klíčová slova
• Old World camels, coronavirus, immune response genes, in-solution hybridization capture, zoonosis,
• MeSH
• adaptivní imunita genetika   MeSH
• bronchy cytologie   fyziologie   MeSH
• cilie fyziologie   MeSH
• COVID-19 genetika   imunologie   virologie   MeSH
• genetická predispozice k nemoci MeSH
• interakce mikroorganismu a hostitele genetika   imunologie   MeSH
• koronavirové infekce genetika   imunologie   přenos   virologie   MeSH
• koronavirus MERS imunologie   izolace a purifikace   patogenita   MeSH
• lidé MeSH
• objevující se infekční nemoci genetika   imunologie   přenos   virologie   MeSH
• přirozená imunita genetika   MeSH
• protilátky virové MeSH
• replikace viru genetika   imunologie   MeSH
• respirační sliznice cytologie   fyziologie   MeSH
• SARS-CoV-2 imunologie   patogenita   MeSH
• velbloudi genetika   imunologie   virologie   MeSH
• zdroje nemoci virologie   MeSH
• zoonózy genetika   imunologie   přenos   virologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Spojené arabské emiráty MeSH
• Názvy látek
• protilátky virové MeSH

BACKGROUND: Immune-response (IR) genes have an important role in the defense against highly variable pathogens, and therefore, diversity in these genomic regions is essential for species' survival and adaptation. Although current genome assemblies from Old World camelids are very useful for investigating genome-wide diversity, demography and population structure, they have inconsistencies and gaps that limit analyses at local genomic scales. Improved and more accurate genome assemblies and annotations are needed to study complex genomic regions like adaptive and innate IR genes. RESULTS: In this work, we improved the genome assemblies of the three Old World camel species - domestic dromedary and Bactrian camel, and the two-humped wild camel - via different computational methods. The newly annotated dromedary genome assembly CamDro3 served as reference to scaffold the NCBI RefSeq genomes of domestic Bactrian and wild camels. These upgraded assemblies were then used to assess nucleotide diversity of IR genes within and between species, and to compare the diversity found in immune genes and the rest of the genes in the genome. We detected differences in the nucleotide diversity among the three Old World camelid species and between IR gene groups, i.e., innate versus adaptive. Among the three species, domestic Bactrian camels showed the highest mean nucleotide diversity. Among the functionally different IR gene groups, the highest mean nucleotide diversity was observed in the major histocompatibility complex. CONCLUSIONS: The new camel genome assemblies were greatly improved in terms of contiguity and increased size with fewer scaffolds, which is of general value for the scientific community. This allowed us to perform in-depth studies on genetic diversity in immunity-related regions of the genome. Our results suggest that differences of diversity across classes of genes appear compatible with a combined role of population history and differential exposures to pathogens, and consequent different selective pressures.

• Klíčová slova
• Chromosome conformation capture, Chromosome mapping, Dromedary, Genetic diversity, Genome annotation, Genome assembly, Immune response genes, Scaffolding,
• MeSH
• anotace sekvence MeSH
• imunoproteiny genetika   MeSH
• jednonukleotidový polymorfismus * MeSH
• kontigové mapování MeSH
• lokus kvantitativního znaku MeSH
• velbloudi genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• imunoproteiny MeSH

The adaptive immune receptors repertoire is highly plastic, with its ability to produce antigen-binding molecules and select those with high affinity for their antigen. Species have developed diverse genetic and structural strategies to create their respective repertoires required for their survival in the different environments. Camelids, until now, considered as a case of evolutionary innovation because of their only heavy-chain antibodies, represent a new mammalian model particularly useful for understanding the role of diversity in the immune system function. Here, we review the structural and functional characteristics and the current status of the genomic organization of camel immunoglobulins (IG) or antibodies, α/ß and γ/δ T cell receptors (TR), and major histocompatibility complex (MHC). In camelid humoral response, in addition to the conventional antibodies, there are IG with "only-heavy-chain" (no light chain, and two identical heavy gamma chains lacking CH1 and with a VH domain designated as VHH). The unique features of these VHH offer advantages in biotechnology and for clinical applications. The TRG and TRD rearranged variable domains of Camelus dromedarius (Arabian camel) display somatic hypermutation (SHM), increasing the intrinsic structural stability in the γ/δ heterodimer and influencing the affinity maturation to a given antigen similar to immunoglobulin genes. The SHM increases the dromedary γ/δ repertoire diversity. In Camelus genus, the general structural organization of the TRB locus is similar to that of the other artiodactyl species, with a pool of TRBV genes positioned at the 5' end of three in tandem D-J-C clusters, followed by a single TRBV gene with an inverted transcriptional orientation located at the 3' end. At the difference of TRG and TRD, the diversity of the TRB variable domains is not shaped by SHM and depends from the classical combinatorial and junctional diversity. The MHC locus is located on chromosome 20 in Camelus dromedarius. Cytogenetic and comparative whole genome analyses revealed the order of the three major regions "Centromere-ClassII-ClassIII-ClassI". Unexpectedly low extent of polymorphisms and haplotypes was observed in all Old World camels despite different geographic origins.

• Klíčová slova
• Camelus bactrianus, Camelus dromedarius, Camelus ferus, Immunoglobulins, Immunome, Old World camelids, T cell receptors, major histocompatibility complex,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

This study brings new information on major histocompatibility complex (MHC) class III sub-region genes in Old World camels and integrates current knowledge of the MHC region into a comprehensive overview for Old World camels. Out of the MHC class III genes characterized, TNFA and the LY6 gene family showed high levels of conservation, characteristic for MHC class III loci in general. For comparison, an MHC class II gene TAP1, not coding for antigen presenting molecules but functionally related to MHC antigen presenting functions was studied. TAP1 had many SNPs, even higher than the MHC class I and II genes encoding antigen presenting molecules. Based on this knowledge and using new camel genomic resources, we constructed an improved genomic map of the entire MHC region of Old World camels. The MHC class III sub-region shows a standard organization similar to that of pig or cattle. The overall genomic structure of the camel MHC is more similar to pig MHC than to cattle MHC. This conclusion is supported by differences in the organization of the MHC class II sub-region, absence of functional DY genes, different organization of MIC genes in the MHC class I sub-region, and generally closer evolutionary relationships of camel and porcine MHC gene sequences analyzed so far.

• Klíčová slova
• Bactrian camel, MHC, Old World camels, SNP, camels, dromedary, major histocompatibility complex,
• MeSH
• antigeny Ly klasifikace   genetika   MeSH
• fylogeneze MeSH
• hlavní histokompatibilní komplex * MeSH
• jednonukleotidový polymorfismus MeSH
• prasata MeSH
• skot MeSH
• TNF-alfa klasifikace   MeSH
• velbloudi genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny Ly MeSH
• TNF-alfa MeSH

Due to production of special homodimeric heavy chain antibodies, somatic hypermutation of their T-cell receptor genes and unusually low diversity of their major histocompatibility complex genes, camels represent an important model for immunogenetic studies. Here, we analyzed genes encoding selected natural killer cell receptors with a special focus on genes encoding receptors for major histocompatibility complex (MHC) class I ligands in the two domestic camel species, Camelus dromedarius and Camelus bactrianus. Based on the dromedary genome assembly CamDro2, we characterized the genetic contents, organization, and variability of two complex genomic regions, the leukocyte receptor complex and the natural killer complex, along with the natural cytotoxicity receptor genes NCR1, NCR2, and NCR3. The genomic organization of the natural killer complex region of camels differs from cattle, the phylogenetically most closely related species. With its minimal set of KLR genes, it resembles this complex in the domestic pig. Similarly, the leukocyte receptor complex of camels is strikingly different from its cattle counterpart. With KIR pseudogenes and few LILR genes, it seems to be simpler than in the pig. The syntenies and protein sequences of the NCR1, NCR2, and NCR3 genes in the dromedary suggest that they could be human orthologues. However, only NCR1 and NCR2 have a structure of functional genes, while NCR3 appears to be a pseudogene. High sequence similarities between the two camel species as well as with the alpaca Vicugna pacos were observed. The polymorphism in all genes analyzed seems to be generally low, similar to the rest of the camel genomes. This first report on natural killer cell receptor genes in camelids adds new data to our understanding of specificities of the camel immune system and its functions, extends our genetic knowledge of the innate immune variation in dromedaries and Bactrian camels, and contributes to studies of natural killer cell receptors evolution in mammals.

• Klíčová slova
• SNP, camelid, leukocyte receptor complex, microsatellites, natural killer complex,
• Publikační typ
• časopisecké články MeSH