Recombinant Bovine ORM1-like protein 1 (ORMDL1)

What is the evolutionary conservation of ORMDL1 across species?

ORMDL1 belongs to a highly conserved gene family (ORMDL) found across diverse organisms. Homologs have been identified in yeast, microsporidia (including opportunistic pathogen Encephalitozoon cuniculi), plants (including Arabidopsis), invertebrates (Drosophila), urochordates (Ciona intestinalis), and various vertebrates. In vertebrates, three distinct ORMDL genes exist (ORMDL1, ORMDL2, and ORMDL3), while Saccharomyces cerevisiae and Arabidopsis thaliana each contain two copies. The high conservation across evolutionary distant species suggests fundamental biological importance of these proteins. Sequence comparisons reveal 80-84% positional identities between human ORMDL proteins, with 116 of 153 amino acid residues being conserved across all three human homologs . This remarkable conservation makes bovine ORMDL1 valuable for comparative studies against human models.

What is the cellular localization and basic function of ORMDL1 proteins?

ORMDL1 encodes a transmembrane protein that is anchored in the endoplasmic reticulum (ER). This localization is consistent across species, indicating conserved subcellular function. Experimental evidence from knockout studies in Saccharomyces cerevisiae demonstrates that deletion of both yeast ORMDL homologs results in decreased growth rates and increased sensitivity to ER stressors like tunicamycin and dithiothreitol . Functional complementation experiments demonstrate that human ORMDL homologs, including ORMDL1, can rescue these yeast mutant phenotypes, confirming functional conservation. The protein participates in ER homeostasis and potentially stress response pathways, which makes it relevant for studies on cellular stress management in bovine cells.

How is ORMDL1 typically expressed in normal bovine tissues?

While the search results don't provide specific data on bovine tissue expression patterns, studies in humans indicate that ORMDL genes are expressed ubiquitously across adult and fetal tissues . This suggests that bovine ORMDL1 likely follows a similar broad expression pattern. Recent research shows ORMDL proteins serve as regulatory subunits for serine palmitoyltransferase (SPT), an enzyme critical for sphingolipid biosynthesis . When studying recombinant bovine ORMDL1, researchers should anticipate expression in virtually all tissue types, with potential quantitative differences that might reflect tissue-specific sphingolipid requirements. Experimental approaches using quantitative PCR or Western blot analysis across bovine tissue panels would be valuable for establishing baseline expression patterns specific to bovine models.

What are the optimal expression systems for producing recombinant bovine ORMDL1?

Based on available research protocols, Escherichia coli expression systems have proven effective for recombinant ORMDL1 production. For bovine ORMDL1 specifically, E. coli-based expression has yielded proteins with greater than 95% purity . The methodological approach typically involves:

• Cloning the bovine ORMDL1 coding sequence (excluding signal peptide) into a bacterial expression vector with appropriate tags (His or GST tags are common)

• Transforming expression-optimized E. coli strains

• Inducing expression under controlled temperature conditions (typically 25-30°C to reduce inclusion body formation)

• Purifying using affinity chromatography appropriate to the chosen tag

For functional studies requiring proper protein folding and post-translational modifications, mammalian expression systems (such as HEK293 or CHO cells) may be preferable, though these are more technically demanding and lower-yielding. When eukaryotic glycosylation patterns are critical, insect cell systems using baculovirus vectors offer a middle-ground approach between bacterial yield and mammalian authenticity.

What purification strategies yield highest quality recombinant bovine ORMDL1?

For high-purity (>95%) recombinant bovine ORMDL1 suitable for functional and structural studies, a multi-step purification protocol is recommended:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins or glutathione-sepharose for GST-tagged proteins)

• Buffer optimization: PBS at pH 7.4 containing protective additives (0.01% SKL, 1mM DTT, 5% trehalose) has shown excellent results

• Secondary purification using size exclusion chromatography to remove aggregates and contaminants

• Quality control via SDS-PAGE and Western blotting to confirm identity and purity

Research indicates that including reducing agents like DTT in purification buffers improves stability, though researchers should note these are classified as hazardous substances requiring appropriate handling . For applications requiring absolute purity, additional ion exchange chromatography steps may be incorporated after initial affinity purification.

How should recombinant bovine ORMDL1 be stored to maintain stability and activity?

Based on established protocols for recombinant ORMDL1 proteins, the following storage conditions maximize stability and preserve functional activity:

• Short-term storage (up to one month): 2-8°C in PBS buffer containing stabilizers

• Long-term storage: -80°C following flash-freezing in suitable cryoprotectants

• Buffer composition: PBS (pH 7.4) containing 0.01% SKL, 1 mM DTT, 5% trehalose, and small amounts of ProClin as preservative

The addition of trehalose significantly enhances freeze-thaw stability by preventing protein aggregation. For applications requiring multiple freeze-thaw cycles, aliquoting prior to freezing is strongly recommended. When preparing working solutions, researchers should avoid repeated freeze-thaw cycles and maintain the protein in reducing conditions to prevent disulfide-mediated aggregation.

How do bovine ORMDL1 knockout models compare to other species in phenotypic presentation?

CRISPR-based knockout approaches have been instrumental in studying ORMDL functions across species. In yeast, double knockout of ORMDL homologs results in significantly decreased growth rates and heightened sensitivity to ER stress inducers like tunicamycin . The generation of mammalian ORMDL knockouts has been achieved using CRISPR-Cas9 technology targeting specific ORMDL isoforms .

When planning bovine ORMDL1 knockout studies, researchers should anticipate potential phenotypes affecting:

• Growth rate and cellular proliferation

• ER stress response pathways

• Sphingolipid metabolism and homeostasis

• Cell membrane composition and signaling

Unlike single ORMDL knockouts, double and triple ORMDL knockout models typically show more pronounced phenotypes due to functional redundancy between ORMDL isoforms. The methodology for generating such models typically involves designing sgRNAs targeting conserved exonic regions, followed by lentiviral delivery of CRISPR components. Viral concentration protocols using polyethylene glycol precipitation have proven effective, with final viral particles being stored in 2% FBS-containing Opti-MEM at -80°C .

What is the specific role of ORMDL1 in regulating serine palmitoyltransferase (SPT) activity?

Recent research has revealed ORMDL proteins, including ORMDL1, function as regulatory subunits of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid biosynthesis . While all three ORMDL isoforms (ORMDL1, ORMDL2, and ORMDL3) participate in SPT regulation, they may exert differential effects under various physiological conditions.

When studying bovine ORMDL1's role in SPT regulation, researchers should consider:

• Quantitative differences in inhibitory potency compared to ORMDL2 and ORMDL3

• Tissue-specific expression patterns that may correlate with sphingolipid requirements

• Regulatory mechanisms governing ORMDL1-SPT interactions

• Potential functional redundancy or compensation mechanisms with other ORMDL isoforms

Methodologically, SPT activity assays following ORMDL1 manipulation (overexpression, knockdown, or mutation) provide direct evidence of regulatory effects. Mass spectrometry-based lipidomic approaches allow for comprehensive analysis of downstream sphingolipid metabolites affected by ORMDL1-mediated SPT regulation. Chromatographic separation using a linear gradient to 100% B1 over 1.5 minutes, held at 100% B1 for 5.5 minutes, followed by a 0.5-minute gradient return has shown effective resolution of sphingolipid species .

What is the current evidence for ORMDL1's role in disease pathogenesis?

While most ORMDL research has focused on ORMDL3's association with asthma, emerging evidence suggests ORMDL1 may have significant roles in cancer biology. Expression analysis using the Gene Expression Profiling Interactive Analysis (GEPIA) database has revealed differential expression of ORMDL1 between normal and tumor tissues across multiple cancer types .

Functional analyses indicate ORMDL1-coexpressed genes in DLBCL participate in critical cellular processes:

These findings suggest ORMDL1 may influence cancer progression through effects on cell cycle control and DNA damage responses . For researchers investigating bovine ORMDL1 in disease contexts, these pathways warrant particular attention, as they may represent conserved mechanisms across species.

How can researchers address solubility challenges with recombinant bovine ORMDL1?

As transmembrane ER-resident proteins, ORMDL family proteins present inherent solubility challenges during recombinant expression. Successfully expressing soluble bovine ORMDL1 requires strategic approaches:

• Tag selection: N-terminal tags (particularly His and GST) have demonstrated effectiveness in improving solubility while maintaining function

• Expression optimization: Lower induction temperatures (16-18°C) and reduced IPTG concentrations often yield higher proportions of soluble protein

• Detergent screening: For functional studies requiring intact transmembrane domains, mild non-ionic detergents (DDM, CHAPS, or digitonin) may preserve native conformation

• Fusion partners: Solubility-enhancing fusion partners like MBP or SUMO can dramatically improve yield of soluble protein

When designing expression constructs, researchers should consider excluding predicted transmembrane regions for applications not requiring membrane integration. For bovine ORMDL1, expression constructs typically include amino acids 19-201 or similar ranges, excluding signal sequences and problematic hydrophobic regions . For structural studies requiring full-length protein, insect cell expression systems may provide better yields of properly folded protein than bacterial systems.

What are the recommended controls for functional studies of recombinant bovine ORMDL1?

Robust controls are essential for meaningful functional characterization of recombinant bovine ORMDL1:

• Expression controls:

  • Empty vector controls processed identically to ORMDL1-expressing systems

  • Related protein controls (non-ORMDL ER transmembrane proteins) to distinguish general from ORMDL-specific effects

• Activity controls:

  • Comparison with human and other mammalian ORMDL1 orthologs to assess functional conservation

  • Parallel analysis of ORMDL2 and ORMDL3 to identify isoform-specific functions

  • Site-directed mutants targeting conserved residues to confirm structure-function relationships

• Rescue experiments:

  • Complementation of yeast ORM knockout phenotypes serves as a gold-standard functional verification

  • Re-expression in CRISPR-generated ORMDL1 knockout mammalian cells

The yeast rescue approach is particularly valuable as double knockouts of yeast ORM homologs show clear phenotypes that can be rescued by human ORMDL homologs . This cross-species complementation provides strong evidence for functional conservation and activity of the recombinant protein.

How can researchers distinguish between the functional contributions of ORMDL1 versus other ORMDL isoforms?

Disentangling the specific functions of ORMDL1 from its homologs ORMDL2 and ORMDL3 presents a significant challenge due to their high sequence similarity (80-84% identity) and potential functional redundancy. Several methodological approaches can help isolate ORMDL1-specific functions:

• Isoform-specific knockdown/knockout:

  • CRISPR-Cas9 targeting unique regions of ORMDL1

  • siRNA/shRNA designed against UTRs or divergent coding regions

  • Sequential knockout of multiple ORMDLs to reveal compensatory mechanisms

• Rescue experiments with specificity controls:

  • Rescue of ORMDL1 knockout with each ORMDL isoform separately

  • Structure-function analysis using chimeric ORMDL proteins

• Tissue and condition-specific expression analysis:

  • Comparative expression profiling across tissues and developmental stages

  • Response to stressors and signaling pathways that might differentially regulate ORMDL isoforms

• Protein interaction studies:

  • Immunoprecipitation coupled with mass spectrometry to identify ORMDL1-specific binding partners

  • Proximity labeling approaches (BioID, APEX) to map isoform-specific interactomes

Generation of CRISPR-based double and triple ORMDL knockout cell lines provides a powerful system for dissecting isoform-specific functions through complementation studies . Similarly, specific antibodies recognizing unique epitopes of ORMDL1 rather than conserved regions enable precise tracking of this isoform distinct from ORMDL2 and ORMDL3.

What emerging technologies might enhance our understanding of bovine ORMDL1 function?

Several cutting-edge technologies have potential to significantly advance bovine ORMDL1 research:

• CRISPR-based approaches:

  • Base editing and prime editing for precise mutation introduction without double-strand breaks

  • CRISPRi/CRISPRa for reversible modulation of ORMDL1 expression

  • CRISPR screens to identify genetic interactors of ORMDL1

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize ORMDL1 distribution within the ER at nanoscale resolution

  • Live-cell imaging with split fluorescent proteins to monitor dynamic protein interactions

  • Correlative light and electron microscopy to connect ORMDL1 localization with ultrastructural features

• Structural biology approaches:

  • Cryo-EM analysis of ORMDL1 in complex with SPT and other partners

  • Hydrogen-deuterium exchange mass spectrometry to map protein interaction surfaces

  • AlphaFold2-guided structural predictions to inform functional studies

• Single-cell technologies:

  • scRNA-seq to reveal cell population-specific ORMDL1 expression patterns

  • Spatial transcriptomics to map ORMDL1 expression in tissue contexts

  • Single-cell proteomics to correlate ORMDL1 levels with cellular phenotypes

These technologies could provide unprecedented insights into how ORMDL1 functions within the complex cellular environment and its specific contributions to sphingolipid metabolism regulation distinct from other ORMDL isoforms.

How might bovine ORMDL1 research translate to therapeutic applications?

While direct therapeutic applications of bovine ORMDL1 research might seem distant, several translational pathways show promise:

• Cancer therapeutics:

  • The association between ORMDL1 expression and poor prognosis in DLBCL suggests potential as a therapeutic target

  • Understanding ORMDL1's role in cell cycle and DNA damage response pathways could inform development of sensitizers to existing chemotherapies

• Inflammatory and metabolic disorders:

  • ORMDL proteins' role in sphingolipid metabolism connects them to inflammatory signaling pathways

  • Selective modulation of ORMDL1 versus other isoforms might offer targeted approaches to diseases involving sphingolipid dysregulation

• Agricultural applications:

  • Insights from bovine ORMDL1 could inform strategies for improving livestock health

  • Understanding species-specific differences in ORMDL function might explain differential susceptibility to certain diseases

Research focused on identifying small molecules that selectively modulate ORMDL1 function could provide valuable tool compounds for proof-of-concept studies. Additionally, comparative studies between human and bovine ORMDL1 might reveal insights applicable to both veterinary and human medicine.

What are the key unanswered questions regarding ORMDL1 biological functions?

Despite growing understanding of ORMDL proteins, several fundamental questions about ORMDL1 remain unanswered:

• Isoform-specific functions:

  • Do the three ORMDL isoforms have distinct biological roles beyond SPT regulation?

  • What mechanisms determine which ORMDL isoform predominates in different cellular contexts?

• Regulatory mechanisms:

  • How is ORMDL1 expression and activity regulated at transcriptional, translational, and post-translational levels?

  • What signaling pathways modulate ORMDL1 function in response to cellular stress or metabolic changes?

• Evolutionary significance:

  • Why have multiple ORMDL isoforms been maintained throughout vertebrate evolution?

  • Do the slight sequence differences between isoforms confer specialized functions?

• Disease relevance:

  • Beyond DLBCL, what other pathological conditions might involve ORMDL1 dysregulation?

  • Could ORMDL1 serve as a biomarker or therapeutic target in specific diseases?

Addressing these questions will require integrated approaches combining genetic, biochemical, and systems biology methodologies. Comparative studies across species, including bovine models, may provide unique insights into conserved and divergent functions of this evolutionarily ancient protein family.