Recombinant Kluyveromyces lactis Protein SEY1 (SEY1)

What is Recombinant Kluyveromyces lactis Protein SEY1?

Recombinant Kluyveromyces lactis Protein SEY1 (SEY1) is a full-length protein (786 amino acids) derived from the yeast K. lactis. It is typically expressed in heterologous systems such as E. coli with an N-terminal His tag to facilitate purification and downstream applications . SEY1 is encoded by the SEY1 gene (KLLA0F20317g) in K. lactis and represents a protein that may be involved in cellular membrane dynamics based on its sequence characteristics. The protein contains functional domains suggesting potential GTPase activity (EC= 3.6.5.-), which may indicate a role in cellular trafficking or membrane fusion events . In research contexts, recombinant SEY1 is primarily used to study yeast cellular processes and as a component in heterologous protein expression systems.

What is the structural composition and physical properties of SEY1 protein?

The SEY1 protein from Kluyveromyces lactis is a 786 amino acid polypeptide with a complex structural organization. The complete amino acid sequence (provided in the product specifications) reveals several conserved domains characteristic of proteins involved in membrane dynamics and cellular trafficking . Physical analysis suggests the protein contains hydrophobic regions that may facilitate membrane association, as well as potential GTPase domains indicated by its EC classification (3.6.5.-) .

When expressed recombinantly with an N-terminal His tag, the protein typically appears as a single band of approximately 87-90 kDa on SDS-PAGE gels, with purity generally exceeding 90% . The protein in its recombinant form is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0, and exhibits optimal stability when stored with 50% glycerol at -20°C or -80°C . Its functional activity, including potential GTPase activity, would need to be assessed through specific enzymatic assays not detailed in the current literature.

How should recombinant SEY1 protein be handled and stored for optimal stability?

Optimal handling and storage of recombinant K. lactis SEY1 protein requires careful attention to temperature, buffer conditions, and freeze-thaw cycles. The protein is typically supplied as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 50% and store aliquots at -20°C or -80°C .

The reconstituted protein should be briefly centrifuged prior to opening to bring contents to the bottom of the vial. Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they significantly compromise protein integrity and activity . The storage buffer typically consists of a Tris/PBS-based solution with 6% trehalose at pH 8.0, which helps maintain protein stability . For researchers conducting functional studies, it's advisable to add protease inhibitors to prevent degradation when working with the protein for extended periods at temperatures above 4°C.

How does oxidative stress affect protein secretion pathways in K. lactis and what implications might this have for SEY1 function?

Oxidative stress significantly impacts protein secretion in K. lactis through multiple mechanisms affecting ER function and secretory pathway efficiency. Research has demonstrated that sustained endoplasmic reticulum stress during recombinant protein production induces oxidative stress in yeast cells, creating a cycle that can further impair proper protein folding and secretion . The accumulation of reactive oxygen species (ROS) during heterologous protein expression can damage cellular components including proteins involved in secretory pathways.

Experimental evidence shows that enhancing antioxidant defenses through overexpression of K. lactis SOD1 (KlSOD1), a superoxide dismutase that catalyzes the dismutation of superoxide anions into oxygen and hydrogen peroxide, significantly improves the production of heterologous proteins such as human serum albumin (HSA) and glucoamylase . The overexpression of KlSOD1 leads to decreased ROS levels during protein production, suggesting that oxidative stress is a limiting factor in efficient protein secretion .

Given that SEY1 may function in membrane dynamics and potentially in the secretory pathway, its activity could be modulated by oxidative stress conditions. Membrane proteins and proteins involved in vesicular trafficking are particularly susceptible to oxidative damage due to their proximity to membrane lipids, which are primary targets for peroxidation. Therefore, SEY1 function might be preserved under conditions where antioxidant defenses are enhanced, potentially contributing to improved secretory capacity. Research investigating the relationship between SEY1 activity and oxidative stress would provide valuable insights into its functional role in the secretory response to cellular stress.

What experimental approaches are most effective for studying SEY1 function in K. lactis?

Multiple complementary experimental approaches are recommended for comprehensive investigation of SEY1 function in K. lactis:

• Gene Disruption/Deletion Analysis: Creating SEY1 knockout strains using CRISPR-Cas9 or homologous recombination techniques would allow assessment of phenotypic changes, particularly related to secretory pathway efficiency and cellular stress responses. Similar approaches with SEL1 revealed a super-secretion phenotype in knockout strains, increasing heterologous protein secretion by five-fold .

• Overexpression Systems: Developing strains with controlled overexpression of SEY1 can reveal gain-of-function phenotypes. This approach has proven effective with other K. lactis genes like SOD1, where overexpression significantly enhanced heterologous protein production .

• Protein Localization Studies: Using fluorescent protein tagging (GFP fusion constructs) to track SEY1 localization within cellular compartments during different growth phases and stress conditions.

• Protein-Protein Interaction Analysis: Employing co-immunoprecipitation, yeast two-hybrid assays, or proximity labeling techniques to identify SEY1 interaction partners within the secretory pathway.

• Biochemical Assays: Testing the predicted GTPase activity (EC 3.6.5.-) of purified recombinant SEY1 protein under various conditions, including oxidative stress.

• Stress Response Experiments: Exposing wild-type and SEY1-modified strains to various stressors, particularly those affecting protein secretion and membrane integrity, would reveal functional relationships. For example, testing strains under oxidative stress conditions (similar to studies with SOD1 ) or ER stress conditions.

• Heterologous Protein Secretion Quantification: Using reporter proteins like human serum albumin (HSA) or bacterial alpha-amylase to quantitatively assess secretory capacity in SEY1-modified strains, as was effectively done in studies of SEL1 and SOD1 .

These approaches should be integrated into a cohesive research program to develop a comprehensive understanding of SEY1's role in K. lactis cellular processes.

What are the optimal expression and purification methods for recombinant K. lactis SEY1 protein?

The optimal expression and purification protocol for recombinant K. lactis SEY1 protein involves several critical steps:

Expression System:
The most effective system documented for SEY1 expression is E. coli with an N-terminal His tag fusion . This approach allows for high-yield production and simplifies purification. The complete coding sequence (encoding amino acids 1-786) should be cloned into an appropriate expression vector with an inducible promoter system (such as T7-based systems).

Culture Conditions:

• Media: LB or TB medium supplemented with appropriate antibiotics

• Induction: IPTG at 0.5-1.0 mM when culture reaches OD600 of 0.6-0.8

• Temperature: Post-induction cultivation at 25-28°C rather than 37°C to enhance proper folding

• Duration: 4-6 hours of induction for optimal yield

Purification Procedure:

• Cell lysis using sonication or pressure-based methods in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Centrifugation at 15,000 × g for 30 minutes to remove cell debris

• Affinity chromatography using Ni-NTA resin with a step gradient of imidazole (10, 50, 250 mM)

• Size exclusion chromatography to enhance purity if necessary

• Buffer exchange to a storage buffer containing Tris/PBS with 6% trehalose, pH 8.0

Quality Control:

• SDS-PAGE analysis to confirm >90% purity

• Western blot using anti-His antibodies to verify identity

• Activity assays to confirm functional integrity, particularly GTPase activity if studying enzymatic function

Storage:
The purified protein should be concentrated to 0.1-1.0 mg/mL, supplemented with glycerol to a final concentration of 50%, aliquoted to minimize freeze-thaw cycles, and stored at -20°C or -80°C for long-term preservation .

This methodology ensures high-quality recombinant SEY1 protein suitable for biochemical, structural, and functional studies.

How can researchers effectively study the relationship between SEY1 and secretory pathway efficiency in K. lactis?

To effectively study the relationship between SEY1 and secretory pathway efficiency in K. lactis, researchers should implement a multi-faceted experimental approach:

1. Genetic Manipulation Strategy:

• Create precise SEY1 knockout strains using CRISPR-Cas9 or homologous recombination

• Develop regulated expression systems (both under- and overexpression) using inducible promoters

• Generate strains with tagged versions of SEY1 (HA, FLAG, or GFP) for localization and interaction studies

2. Heterologous Reporter System:
Establish a quantitative secretion assay using well-characterized reporter proteins:

• Bacillus amyloliquefaciens α-amylase (successfully used in K. lactis secretion studies)

• Human serum albumin (HSA) (demonstrated utility in K. lactis secretion analysis)

• Arxula adeninivorans glucoamylase (proven effective for assessing secretion efficiency)

3. Secretory Pathway Analysis:

• Microscopy techniques to track protein movement through the secretory pathway

• Subcellular fractionation to quantify reporter protein in different compartments

• Glycosylation analysis to assess processing efficiency

4. Stress Response Evaluation:

• Measure ROS levels using fluorescent probes like DCFH-DA in wild-type vs. SEY1-modified strains

• Test the effects of antioxidants (e.g., ascorbic acid) and ROS generators (e.g., menadione) on secretion efficiency in SEY1-modified strains

• Evaluate ER stress markers (e.g., UPR activation) in relation to SEY1 expression levels

5. Interaction Network Mapping:

• Perform co-immunoprecipitation with tagged SEY1 to identify binding partners

• Use proximity labeling techniques (BioID or APEX) to identify transient interactions

• Conduct genetic interaction screens to identify synthetic lethal or synthetic rescue phenotypes

6. Comparative Analysis:
Establish a standardized secretion assay to compare SEY1 function across conditions:

This comprehensive approach would provide mechanistic insights into SEY1's role in the secretory pathway and its potential utility for enhancing heterologous protein production in K. lactis.

What techniques can be used to investigate how oxidative stress affects SEY1 function?

Investigating the relationship between oxidative stress and SEY1 function requires specialized techniques spanning molecular biology, biochemistry, and cell biology approaches:

1. Oxidative Stress Induction and Measurement:

• Chemical induction using menadione (a superoxide generator), hydrogen peroxide, or tert-butyl hydroperoxide at standardized concentrations

• Environmental stress induction through high-aeration cultivation conditions

• ROS quantification using fluorescent probes such as DCFH-DA (for general ROS) or MitoSOX (for mitochondrial superoxide)

• Lipid peroxidation assessment using TBARS (thiobarbituric acid reactive substances) assay

• Protein carbonylation measurement using DNPH (2,4-dinitrophenylhydrazine) derivatization and immunoblotting

2. SEY1 Functional Assessment Under Oxidative Stress:

• In vitro GTPase activity assays with purified recombinant SEY1 protein exposed to varying concentrations of oxidants

• Site-directed mutagenesis of redox-sensitive residues (cysteine, methionine) to identify critical sites for oxidative regulation

• Analysis of post-translational modifications induced by oxidative stress using mass spectrometry

3. Cellular Response and Localization Studies:

• Live-cell imaging with fluorescently tagged SEY1 under normal and oxidative stress conditions

• Co-localization analysis with ER, Golgi, and secretory vesicle markers during oxidative stress

• FRAP (Fluorescence Recovery After Photobleaching) analysis to assess SEY1 mobility under stress conditions

4. Genetic Approaches:

• Expression analysis of SEY1 under oxidative stress using RT-qPCR

• Construction of dual reporter strains expressing both SEY1-GFP and oxidative stress-responsive promoter driving RFP

• Synthetic genetic array analysis combining SEY1 mutations with antioxidant gene deletions

5. Proteomic Interaction Analysis:

• Comparative interactome analysis of SEY1 under normal versus oxidative stress conditions

• Redox proteomics to identify changes in the oxidation state of SEY1 and interacting partners

6. Functional Assessment in Antioxidant-Modulated Systems:
Similar to studies with SOD1 , researchers should:

• Test SEY1 function in strains overexpressing antioxidant enzymes like SOD1

• Assess SEY1-dependent phenotypes in the presence of antioxidants like ascorbic acid

• Compare secretion efficiency in SEY1-modified strains under various oxidative stress conditions

These techniques would provide comprehensive insights into how oxidative stress affects SEY1 structure, localization, interactions, and function, potentially revealing mechanisms to enhance protein secretion through redox modulation of secretory pathway components.

How might SEY1 manipulation be utilized to enhance heterologous protein production in K. lactis?

Based on our understanding of secretory pathway dynamics in yeast, strategic manipulation of SEY1 could potentially enhance heterologous protein production in K. lactis through several mechanisms:

1. Pathway Engineering Approaches:
If SEY1 functions similarly to other secretory pathway components, its expression level could be optimized to enhance secretory capacity. The precedent for this approach comes from studies of SEL1, where gene disruption increased heterologous protein secretion by five-fold . Depending on whether SEY1 acts as a positive or negative regulator of secretion, either overexpression or controlled downregulation strategies might be implemented.

2. Stress Response Integration:
Given the established link between oxidative stress and secretion efficiency, SEY1 manipulation could be combined with antioxidant strategies. Research has demonstrated that SOD1 overexpression significantly enhances heterologous protein production while reducing ROS levels . A dual-modification approach combining SEY1 optimization with enhanced antioxidant capacity (through SOD1 overexpression or media supplementation with ascorbic acid) might yield synergistic improvements in secretion efficiency.

3. Conditional Expression Systems:
Development of inducible or growth phase-specific SEY1 expression systems could allow dynamic regulation of secretory pathway capacity. This approach would be particularly valuable if constitutive SEY1 modification negatively impacts cell growth or viability while still benefiting protein secretion.

4. Protein-Specific Optimization:
The optimal SEY1 expression level may vary depending on the heterologous protein being produced. Creating a panel of strains with varying SEY1 expression levels would allow empirical determination of the best host for specific target proteins. This strategy could be particularly valuable for challenging-to-express proteins that tend to aggregate or misfold.

5. Multi-Gene Modification Approach:
Combining SEY1 manipulation with modifications to other secretory pathway genes could address multiple bottlenecks simultaneously. Previous research has identified at least 10 different DNA sequences that can suppress super-secreting phenotypes in K. lactis , suggesting multiple potential targets for combinatorial optimization.

Implementation of these strategies requires further research to characterize SEY1's precise role in K. lactis, but the potential for enhancing industrial protein production makes this a promising direction for future investigation.

What are the critical experimental controls needed when evaluating SEY1 function in heterologous expression systems?

When designing experiments to evaluate SEY1 function in heterologous expression systems, the following critical controls should be incorporated to ensure reliable and interpretable results:

1. Genetic Background Controls:

• Isogenic wild-type strain (differing only in SEY1 status)

• Empty vector controls for overexpression studies

• Complementation controls (reintroduction of wild-type SEY1 into knockout strains)

• Non-targeted CRISPR control for gene editing approaches

2. Protein Expression and Secretion Controls:

• Non-secreted cytoplasmic reporter protein to distinguish secretion effects from general expression effects

• Well-characterized secretory protein with established behavior in K. lactis (e.g., α-amylase)

• Multiple independent transformants to account for clone-to-clone variation

• Time-course measurements to distinguish between secretion rate effects and final yield effects

3. Specificity Controls:

• Manipulation of known secretory pathway genes as positive controls (e.g., SEL1)

• Overexpression of antioxidant genes (e.g., SOD1) as comparative controls for secretion enhancement

• Testing multiple heterologous proteins with different properties (e.g., HSA and glucoamylase)

4. Stress Response Controls:

• Oxidative stress controls: cells treated with antioxidants (ascorbic acid) or oxidants (menadione)

• Growth controls under various conditions (standard media, high osmolarity, different carbon sources)

• Measurement of stress markers (ROS levels, UPR activation) alongside secretion measurement

5. Technical Controls:

• Loading controls for western blot analysis

• Standard curves for quantitative assays

• Multiple biological and technical replicates

• Media-only controls to establish baseline for secreted protein measurements

6. Systematic Data Collection:
Comprehensive data collection across multiple parameters allows for proper interpretation:

These controls ensure that any observed effects can be specifically attributed to SEY1 function rather than experimental artifacts or general stress responses, providing a solid foundation for understanding SEY1's role in heterologous protein expression systems.

What are the key unresolved questions regarding K. lactis SEY1 function?

Despite the available information on recombinant K. lactis SEY1 protein, several critical questions remain unresolved that represent important opportunities for future research:

• Precise Molecular Function: While sequence analysis suggests GTPase activity (EC 3.6.5.-) , the specific substrates, regulators, and kinetic properties of SEY1 remain largely uncharacterized. Detailed biochemical studies are needed to establish its enzymatic parameters and regulation mechanisms.

• Subcellular Localization and Trafficking: The dynamic localization of SEY1 throughout the cell cycle and under various stress conditions remains undefined. Understanding its movement between cellular compartments would provide insights into its functional roles.

• Interaction Network: The protein-protein interaction network surrounding SEY1 is largely unmapped. Identifying binding partners would clarify its position within cellular pathways and potentially reveal novel regulatory mechanisms.

• Regulatory Mechanisms: How SEY1 expression and activity are regulated at transcriptional, translational, and post-translational levels remains unexplored. This includes potential regulation in response to secretory pathway stress or oxidative stress.

• Comparative Function Across Species: While the SEY1 protein is conserved across yeast species, the degree of functional conservation and species-specific adaptations remain unclear. Cross-species complementation studies could address this gap.

• Relationship to Secretory Efficiency: Unlike SEL1, whose disruption enhances secretion , or SOD1, whose overexpression improves heterologous protein production , the impact of SEY1 manipulation on secretory capacity has not been systematically evaluated.

• Structural Characterization: High-resolution structural data (crystal structure or cryo-EM) for SEY1 would provide valuable insights into its functional domains and mechanism of action.

These unresolved questions represent significant opportunities for researchers to advance our understanding of yeast secretory pathways and potentially develop improved systems for heterologous protein production.

How do findings about K. lactis SEY1 contribute to broader understanding of yeast secretory pathways?

Research on K. lactis SEY1 contributes to our broader understanding of yeast secretory pathways in several significant ways, integrating with existing knowledge about protein secretion mechanisms:

• Evolutionary Conservation and Divergence: Studying SEY1 in K. lactis provides insights into the evolutionary conservation of secretory pathway components across yeast species. The functional complementation observed with other secretory pathway genes like SEL1 between S. cerevisiae and K. lactis suggests conserved core machinery with species-specific adaptations . This comparative perspective helps identify both fundamental and specialized secretory mechanisms.

• Integrated Stress Responses: The connection between oxidative stress and secretion efficiency revealed by SOD1 studies in K. lactis demonstrates how cellular stress response systems are integrated with secretory function . This suggests that secretory pathways do not operate in isolation but are influenced by multiple cellular systems, potentially including SEY1-mediated processes.

• Multi-Level Regulation: The identification of multiple genes that can suppress super-secretion phenotypes in K. lactis (including SEL1) indicates that secretory efficiency is regulated at multiple levels . Understanding SEY1's position within this regulatory network would further illuminate the complex control mechanisms governing secretion.

• Biotechnological Applications: K. lactis has emerged as an important alternative to S. cerevisiae for heterologous protein production, particularly for proteins intended for pharmaceutical or food applications. Characterizing SEY1's role would contribute to rational strain engineering approaches aimed at optimizing secretory capacity.

• Alternative Secretory Pathways: Research in various yeast species has revealed the existence of both conventional and unconventional secretory pathways. Determining whether SEY1 functions in standard ER-Golgi trafficking or in alternative secretory routes would enhance our understanding of the diversity of secretory mechanisms.