Recombinant UPF0299 membrane protein VP1300 (VP1300)

Expression Systems for VP1300

Q: What expression system is most commonly used for recombinant UPF0299 membrane protein VP1300?

Protein Sequence and Features

Q: What is the amino acid sequence and key structural features of the VP1300 protein?

A: The full amino acid sequence of UPF0299 membrane protein VP1300 is: "MIKDRFLQLIQLLISLFLIMGALGIGITIQKFTGVSVPGSVIGMLVLFFSMTLGLVKVDWVKPGATLFIRYMILLFVPISVGLMQHFDMLLANALPIIASAVGGSLIVLVSLAWLLDYLLKEKH" . This 124-amino acid protein is classified as a membrane protein, suggesting it contains hydrophobic regions that integrate into lipid bilayers. Analysis of the sequence reveals characteristic features of membrane proteins, including hydrophobic stretches that likely form transmembrane domains. The protein's membrane localization makes it challenging to work with using standard protein techniques, requiring specialized solubilization and purification approaches to maintain its native structure and function during experimental procedures.

Storage and Stability Considerations

Q: How should recombinant VP1300 protein be stored to maintain stability?

A: For optimal stability of recombinant VP1300 protein, it is recommended to store the lyophilized powder at -20°C/-80°C upon receipt . For working solutions, aliquoting is necessary to avoid repeated freeze-thaw cycles, which can significantly reduce protein stability and activity . The recommended storage buffer typically consists of a Tris-based buffer with 50% glycerol optimized for this protein . Alternatively, a Tris/PBS-based buffer with 6% trehalose at pH 8.0 has also been used . For short-term use, working aliquots can be stored at 4°C for up to one week . If reconstituting from lyophilized form, it's advised to briefly centrifuge the vial before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (final concentration) for long-term storage .

Reconstitution Protocol

Q: What is the recommended protocol for reconstituting lyophilized VP1300 protein?

A: The recommended reconstitution protocol for lyophilized VP1300 protein begins with a brief centrifugation of the vial prior to opening to ensure the protein powder is at the bottom of the container . The protein should then be reconstituted in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL . For long-term storage stability, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being commonly used) . After reconstitution, the solution should be gently mixed until completely dissolved, avoiding vigorous shaking or vortexing which could denature the membrane protein. The reconstituted protein solution should then be aliquoted into smaller volumes to prevent repeated freeze-thaw cycles and stored at -20°C or -80°C for long-term storage .

Purification Strategies

Q: What are the optimal purification strategies for maintaining the structural integrity of VP1300 during isolation?

A: Purifying membrane proteins like VP1300 while maintaining their structural integrity requires specialized approaches. For VP1300, a His-tagged purification strategy has proven successful, but several critical considerations must be addressed . Initially, the protein is often found in insoluble fractions after expression, necessitating solubilization with detergents or chaotropic agents like urea . Based on established membrane protein purification methods, a recommended approach would involve cell lysis under native conditions, followed by membrane fraction isolation through ultracentrifugation . The membrane fraction can then be solubilized using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) to extract VP1300 while preserving its native structure . Immobilized metal affinity chromatography (IMAC) utilizing the His-tag can be employed for initial purification, followed by size-exclusion chromatography to ensure high purity and structural homogeneity . Throughout the purification process, protein quality can be monitored using techniques like fluorescence-detected size exclusion chromatography (FSEC), which allows assessment of protein folding and monodispersity without requiring large amounts of purified protein .

Functional Characterization

Q: What experimental approaches can be used to characterize the function of VP1300 in vitro?

A: Characterizing the function of VP1300, a membrane protein with no clearly established function from the search results, requires a multi-faceted experimental approach. Based on methodologies used for similar membrane proteins, researchers should consider a combination of biophysical and biochemical techniques. Circular dichroism (CD) spectroscopy can provide information about the protein's secondary structure and stability in different detergent environments . For membrane integration studies, a fluorescence-based liposome reconstitution assay would determine if the purified protein can successfully incorporate into artificial membranes and maintain its native conformation . Given VP1300's bacterial origin from Vibrio parahaemolyticus, comparative genomics and protein interaction studies using pull-down assays with potential partner proteins from related pathways could provide functional insights . Additionally, site-directed mutagenesis of conserved residues followed by functional assays would help identify critical regions for activity. For more detailed structural characterization, techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) could map solvent-accessible regions and potential binding interfaces . These complementary approaches would collectively provide valuable insights into VP1300's functional properties despite the limited prior knowledge of its specific biological role.

Expression System Optimization

Q: How can expression systems be optimized to improve the yield and quality of recombinant VP1300?

Structural Studies Considerations

Q: What are the key considerations for conducting structural studies of VP1300 using X-ray crystallography?

A: Conducting structural studies of VP1300 via X-ray crystallography presents several unique challenges due to its membrane protein nature. Based on established approaches for membrane protein crystallography, researchers should first focus on protein stabilization through systematic screening of detergents, as the choice of detergent critically influences crystallization success . Consider implementing the lipidic cubic phase (LCP) crystallization method, which provides a membrane-like environment conducive to forming well-ordered membrane protein crystals . For VP1300 specifically, engineering approaches may be necessary, such as creating fusion constructs with crystallization chaperones (e.g., T4 lysozyme or BRIL) inserted into flexible loop regions to provide crystal contact points without disrupting the core structure . Thermostability assays (CPM-based thermal shift) should be employed to identify conditions (pH, salt, additives) that maximize protein stability prior to crystallization trials . Screening with antibody fragments (Fab or nanobody) as crystallization chaperones can provide additional crystal contacts and potentially lock the protein in a specific conformation . For data collection, consider using microfocus beamlines at synchrotron facilities specialized for small, weakly-diffracting membrane protein crystals. Finally, molecular replacement using structurally similar membrane proteins may facilitate phase determination, though de novo phasing using selenomethionine or heavy atom derivatives might be necessary given the limited structural information available for VP1300 .

Host-Pathogen Interaction Studies

Q: How can recombinant VP1300 be utilized to study potential roles in host-pathogen interactions of Vibrio parahaemolyticus?

A: Investigating VP1300's potential role in host-pathogen interactions requires a systematic experimental approach. Given that VP1300 originates from Vibrio parahaemolyticus, a foodborne pathogen, researchers should first establish cell culture infection models using relevant human intestinal epithelial cell lines (e.g., Caco-2, HT-29) . Purified recombinant VP1300 can be used in binding assays with these cell lines to determine if the protein directly interacts with host cell receptors or membranes . For more detailed interaction studies, biotinylated VP1300 coupled with pull-down assays followed by mass spectrometry analysis would identify potential host protein binding partners . Competition experiments using anti-VP1300 antibodies during bacterial infection can reveal whether the protein is accessible on the bacterial surface and functionally relevant during infection . To assess immunological aspects, purified VP1300 can be used to stimulate human immune cells (such as macrophages or dendritic cells) while monitoring cytokine production and immune activation markers . For in vivo relevance, constructing VP1300 knockout strains of Vibrio parahaemolyticus and comparing their virulence to wild-type strains in appropriate animal models would definitively establish the protein's role in pathogenesis . Collectively, these approaches would provide comprehensive insights into whether VP1300 contributes to the pathogenicity of Vibrio parahaemolyticus through direct host interactions.

Expression Challenges

Q: What are common challenges encountered when expressing VP1300 and how can they be addressed?

A: Expression of membrane proteins like VP1300 presents several common challenges that require specific troubleshooting approaches. One frequent issue is protein toxicity to the host cells, resulting in poor growth and low yields . This can be addressed by using specialized E. coli strains designed for toxic protein expression (such as C41/C43) or expression systems with tightly controlled inducible promoters . Another common challenge is protein misfolding leading to inclusion body formation. To minimize this, researchers should optimize induction conditions by lowering the temperature (to 16-20°C), reducing inducer concentration, and extending expression time . For VP1300 specifically, inclusion body formation might be unavoidable, in which case refolding protocols using gradual detergent exchange or on-column refolding methods may be necessary . Protein degradation during expression can be combated by adding protease inhibitors during cell lysis and purification, or by co-expressing with molecular chaperones that assist in proper folding . If expression yields remain problematic, consider redesigning the construct with modified N- or C-termini, as terminal residues can significantly impact expression efficiency of membrane proteins . Finally, if E. coli consistently yields poorly folded protein despite optimization, transitioning to eukaryotic expression systems such as insect cells may be necessary, despite the increased complexity and cost .

Purification Problems

Q: What are the main challenges in purifying VP1300 and how can they be overcome?

A: Purifying membrane proteins like VP1300 presents several specific challenges that require systematic troubleshooting. A primary challenge is maintaining protein stability during extraction from membranes. This can be addressed by screening multiple detergents (ranging from harsh ionic detergents like SDS to milder non-ionic ones like DDM or LMNG) to identify optimal solubilization conditions . Protein aggregation during purification is another common issue, which can be monitored using techniques like dynamic light scattering (DLS) or size-exclusion chromatography . To minimize aggregation, maintain consistent cold temperatures throughout purification, add stabilizing agents like glycerol or specific lipids, and optimize buffer conditions (pH, salt concentration) . Low binding affinity to purification resins (particularly for His-tagged VP1300) can be addressed by adjusting imidazole concentrations in binding and wash buffers, extending binding time, or considering alternative tags like FLAG or Strep-II . Protein heterogeneity, which complicates structural studies, can be assessed using fluorescence-detected size exclusion chromatography (FSEC) and potentially improved by adding specific lipids that stabilize the native conformation or by removing flexible regions through limited proteolysis followed by mass spectrometry analysis to design optimized constructs . Finally, consider using amphipols or nanodiscs as alternatives to detergents for maintaining VP1300 in a membrane-like environment during later purification steps and subsequent structural or functional studies .

Protein Solubility Issues

Q: How can the solubility of recombinant VP1300 be improved for functional studies?

A: Improving the solubility of recombinant VP1300 for functional studies requires specific strategies tailored to membrane proteins. One effective approach is to conduct a comprehensive detergent screen, testing both traditional detergents (DDM, LMNG, OG) and newer amphipathic agents such as SMA copolymers or amphipols, which have shown superior ability to maintain membrane protein solubility and function . For VP1300 specifically, consider incorporating lipids that match its native membrane environment during purification and storage, as lipids can critically stabilize membrane protein structure . Fusion partners that enhance solubility, such as MBP (maltose-binding protein) or SUMO, can be engineered at the N-terminus while keeping the His-tag for purification purposes . If the protein remains challenging, implementing directed evolution or alanine-scanning mutagenesis approaches can identify specific residues that, when mutated, enhance solubility without compromising function . Temperature optimization during handling is crucial - maintaining samples at 4°C throughout purification and functional assays can significantly reduce aggregation tendencies . For long-term studies, reconstitution into nanodiscs or liposomes provides a more native-like membrane environment than detergent micelles, potentially preserving functional properties better . Finally, consider adding specific stabilizing agents to your buffers, such as glycerol (up to 10%), specific salt concentrations based on thermostability assays, or cholesterol hemisuccinate for additional membrane protein stabilization .

Current Research Limitations

Q: What are the current research limitations in working with VP1300 and what future directions might address these challenges?

A: Current research on VP1300 faces several significant limitations that affect comprehensive characterization of this membrane protein. Based on available information, there appears to be limited functional annotation of VP1300, hampering hypothesis-driven research into its biological role . The hydrophobic nature of this membrane protein creates persistent technical challenges in obtaining sufficient quantities of properly folded protein for advanced structural and functional studies . Additionally, there is an apparent lack of published structural data for VP1300, including crystal structures or cryo-EM models, which limits molecular-level understanding of its function . Future research directions should address these limitations through several approaches. Implementation of advanced membrane protein crystallization techniques like lipidic cubic phase crystallography or single-particle cryo-EM analysis could provide structural insights . Development of specific antibodies against VP1300 would facilitate cellular localization studies and potential immunoprecipitation experiments to identify interaction partners . Comparative genomic analyses across Vibrio species could help predict functional roles based on conservation patterns and genomic context . For functional characterization, systematic phenotyping of VP1300 knockout strains under various stress conditions would likely reveal physiological roles . Finally, applying emerging technologies like hydrogen-deuterium exchange mass spectrometry (HDX-MS) could provide insights into protein dynamics and potential ligand-binding sites even in the absence of high-resolution structures .

Emerging Research Applications