Recombinant Listeria innocua serovar 6a UPF0316 protein lin1888 (lin1888)

What is Recombinant Listeria innocua serovar 6a UPF0316 protein lin1888?

Recombinant Listeria innocua serovar 6a UPF0316 protein lin1888 is a full-length protein derived from the bacterial species Listeria innocua serovar 6a (strain CLIP 11262). It consists of 174 amino acids and is classified as part of the UPF0316 protein family. The protein is typically produced using recombinant expression systems, most commonly in E. coli hosts, and can be tagged (often with a histidine tag) to facilitate purification and experimental applications. The UniProt accession number for this protein is Q92AN2 . This protein is significant in Listeria research as it represents a non-pathogenic counterpart that can be used in comparative studies with pathogenic Listeria species.

How should recombinant lin1888 protein be stored for optimal stability?

For optimal stability of recombinant lin1888 protein, the following storage conditions are recommended based on established protocols: The purified protein should be stored in a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein. For short-term storage (up to one week), working aliquots can be kept at 4°C. For intermediate storage, maintain the protein at -20°C. For long-term preservation, storage at -80°C is recommended .

It is critical to avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity through denaturation and aggregation. Researchers should prepare multiple small-volume aliquots during the initial purification process to minimize the number of freeze-thaw events. Additionally, when thawing the protein, it should be done gradually on ice rather than at room temperature to prevent protein degradation and maintain structural integrity.

What expression systems are most effective for producing recombinant lin1888?

E. coli expression systems are most commonly employed for the recombinant production of lin1888 protein, as evidenced by commercial preparations . For optimal expression, BL21(DE3) or Rosetta(DE3) strains are recommended due to their reduced protease activity and enhanced expression capabilities for proteins containing rare codons, which can be an issue with bacterial proteins expressed in heterologous systems.

The methodology typically involves cloning the lin1888 gene into an expression vector containing an inducible promoter (such as T7 or tac), with an N-terminal or C-terminal His-tag to facilitate purification. Induction is typically performed using IPTG at concentrations between 0.1-1.0 mM when the culture reaches mid-log phase (OD600 of 0.6-0.8). Expression should be conducted at lower temperatures (16-25°C) rather than the standard 37°C to enhance proper folding and solubility, especially given the multiple transmembrane domains present in this protein. Purification is most effectively achieved using nickel affinity chromatography followed by size exclusion chromatography to obtain a homogeneous preparation.

How does lin1888 protein compare to homologous proteins in pathogenic Listeria species?

When conducting comparative studies, researchers should employ multiple sequence alignment tools such as Clustal Omega or MUSCLE to identify conserved and variable regions. Structural prediction analysis using tools like Phyre2 or I-TASSER can provide insights into potential functional differences. The methodological approach should include not only sequence-based comparisons but also experimental validation through complementation studies, where the lin1888 gene is expressed in L. monocytogenes strains with the corresponding gene knocked out, to assess functional equivalence or divergence .

What experimental approaches are most effective for studying lin1888 protein-protein interactions?

For investigating protein-protein interactions involving lin1888, a multi-faceted approach is recommended due to the membrane-associated nature of this protein:

• Co-immunoprecipitation (Co-IP): Using antibodies against lin1888 or its tagged version (His-tagged) to pull down protein complexes from cell lysates, followed by mass spectrometry identification of binding partners.

• Bacterial Two-Hybrid System: More suitable than yeast two-hybrid for bacterial membrane proteins, this approach can identify direct protein interactions in a bacterial cellular context.

• Proximity-dependent Biotin Identification (BioID): By fusing a biotin ligase to lin1888, proteins in close proximity become biotinylated and can be subsequently identified.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics between purified lin1888 and candidate interacting proteins.

• Microscale Thermophoresis (MST): A solution-based technique that can detect interactions with minimal protein amounts and is suitable for membrane proteins.

When interpreting interaction data, researchers should validate findings through multiple orthogonal techniques and consider the potential impact of the expression system and purification methods on the protein's native conformation and interaction capabilities .

How can lin1888 be used as a surrogate model in comparative Listeria research?

Listeria innocua strains, including those expressing the lin1888 protein, can serve as valuable surrogates for pathogenic Listeria monocytogenes in various research applications. This approach is particularly useful in biosafety level 1 (BSL-1) laboratories where working with pathogenic Listeria species would require higher containment levels. The methodology for employing lin1888 as a surrogate model involves:

• Selection of appropriate L. innocua strains: Research indicates that strain-dependent responses exist, with certain L. innocua strains more closely mimicking the behavior of L. monocytogenes under specific experimental conditions. For inactivation studies, a cocktail of two L. innocua strains (PFR 05A07 and PFR 05A10) has demonstrated responses most similar to those of L. monocytogenes cocktails .

• Validation of surrogate appropriateness: Before extrapolating results, researchers must validate that the selected L. innocua strain(s) behave similarly to L. monocytogenes under the specific experimental conditions being tested. This validation should include:

  • Comparative growth kinetics analysis

  • Stress response profiling

  • Protein expression analysis focusing on the specific proteins of interest, including lin1888

• Application in inactivation studies: When investigating thermal inactivation (55°C), UV-C irradiation (245 nm), or chemical sanitizers (such as peroxyacetic acid-based solutions), researchers should be aware that inactivation plateaus may occur (after approximately 120 minutes for thermal treatment) and that responses are highly strain-dependent .

What analytical techniques are most suitable for characterizing the structure and function of lin1888?

For comprehensive characterization of lin1888 structure and function, researchers should employ a combination of complementary analytical techniques:

When analyzing transmembrane proteins like lin1888, special consideration must be given to the membrane environment. Reconstitution into lipid nanodiscs or liposomes can provide a more native-like environment than detergent-solubilized preparations, potentially yielding more physiologically relevant structural and functional insights .

How does the inactivation profile of bacteria expressing lin1888 compare across different treatment methods?

Studies on Listeria innocua strains, which express lin1888, reveal distinct inactivation profiles across different treatment methods. This information is valuable for both food safety applications and fundamental research into bacterial stress responses. The comparative inactivation methodology and results include:

What are the optimal expression conditions for maximizing soluble lin1888 protein yield?

Maximizing soluble yield of recombinant lin1888 protein requires careful optimization of expression conditions, particularly given its membrane-associated nature. The following methodological approach is recommended:

• Vector Selection: Use a vector with a tightly controlled inducible promoter (T7 or similar) and include a fusion tag that enhances solubility (such as SUMO, MBP, or GST) in addition to the His-tag for purification.

• Expression Parameters:

  • Temperature: Lower expression temperatures (16-18°C) typically enhance proper folding and reduce inclusion body formation

  • Induction OD600: Begin induction at mid-log phase (OD600 = 0.6-0.8)

  • Inducer Concentration: For IPTG, start with a lower concentration (0.1-0.2 mM) to promote slower expression

  • Duration: Extended expression periods (16-20 hours) at lower temperatures often yield more soluble protein

• Media and Additives:

  • Rich media (such as Terrific Broth) often provides better yields than standard LB

  • Consider supplementing with additives that can stabilize membrane proteins:

    • Glycerol (5-10%)

    • Specific ions (Mg2+, Ca2+) at 5-10 mM

    • Mild detergents (0.05-0.1% Triton X-100) during cell lysis

• Cell Lysis and Protein Extraction:

  • For membrane proteins like lin1888, standard sonication or high-pressure homogenization should be followed by membrane fraction isolation through ultracentrifugation

  • Subsequent solubilization using appropriate detergents (DDM, LDAO, or Fos-choline at concentrations just above their CMC) is critical

  • Include protease inhibitors throughout the purification process

By systematically optimizing these parameters through small-scale expression trials before scaling up, researchers can significantly improve the yield of soluble, properly folded lin1888 protein for subsequent structural and functional studies .

What purification strategies are most effective for obtaining high-purity lin1888 for structural studies?

Obtaining high-purity lin1888 protein suitable for structural studies requires a multi-step purification strategy that preserves the native conformation of this membrane protein:

• Initial Affinity Chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary capture step for His-tagged lin1888

  • Use a gradient elution with imidazole (20-500 mM) rather than step elution to improve separation

  • Include the optimal detergent (determined during expression optimization) in all buffers at concentrations above the critical micelle concentration (CMC)

• Intermediate Purification:

  • Ion exchange chromatography (IEX) can remove contaminants with different charge properties

  • For lin1888 with a theoretical pI of approximately 9.3 (based on sequence analysis), cation exchange at neutral pH would be appropriate

  • Alternatively, size exclusion chromatography (SEC) can separate protein aggregates and lower molecular weight contaminants

• Final Polishing:

  • Size exclusion chromatography as the final step ensures monodispersity, which is critical for structural studies

  • Multi-angle light scattering (MALS) coupled with SEC provides information about the oligomeric state and homogeneity

  • Consider using fluorescence-detection size exclusion chromatography (FSEC) to assess protein stability and homogeneity before committing to large-scale purification

• Detergent Exchange or Reconstitution:

  • For structural studies, the detergent may need to be exchanged to one more suitable for the specific technique (e.g., DDM for cryo-EM, shorter chain detergents for crystallization)

  • Alternatively, reconstitution into nanodiscs, liposomes, or amphipols can provide a more native-like environment

• Quality Assessment:

  • SDS-PAGE analysis should show >95% purity

  • Mass spectrometry confirmation of protein identity and integrity

  • Circular dichroism to verify secondary structure integrity

  • Thermal stability assays (DSF or nanoDSF) to assess protein folding and stability

This strategic approach maximizes not only the purity but also the structural integrity and homogeneity of lin1888, which are prerequisite factors for successful structural biology investigations .