Recombinant Sodalis glossinidius UPF0060 membrane protein SG1469 (SG1469)

What is Recombinant Sodalis glossinidius UPF0060 membrane protein SG1469?

Recombinant SG1469 is a full-length UPF0060 family membrane protein derived from the insect endosymbiont Sodalis glossinidius. It consists of 132 amino acids and is typically expressed with an N-terminal His-tag in E. coli expression systems . SG1469 is classified as a multi-pass membrane protein that localizes to the cell inner membrane . The recombinant form allows researchers to study this protein's structure and function outside its native environment, facilitating investigations into its role in symbiotic relationships between bacteria and insect hosts, particularly in the context of tsetse flies where S. glossinidius serves as an important endosymbiont .

How should SG1469 protein be stored and handled in laboratory settings?

For optimal stability and functionality, recombinant SG1469 protein requires specific storage and handling protocols:

Researchers should centrifuge vials briefly before opening to bring contents to the bottom . For lyophilized protein, reconstitution should be performed carefully to ensure complete solubilization while maintaining protein integrity. The shelf life is approximately 6 months for liquid formulations and 12 months for lyophilized preparations when stored at -20°C/-80°C .

What expression systems are optimal for producing recombinant SG1469?

The most effective expression system documented for recombinant SG1469 production is E. coli . When designing expression strategies for this membrane protein, researchers should consider the following methodological approaches:

• Expression vector selection: Vectors with strong, inducible promoters (like T7) and appropriate fusion tags (His-tag) facilitate expression and purification.

• Host strain optimization: E. coli strains optimized for membrane protein expression, such as C41(DE3) or C43(DE3), may improve yields compared to standard BL21(DE3) strains.

• Induction conditions: Optimize IPTG concentration (typically 0.1-1.0 mM), induction temperature (often lowered to 16-25°C), and induction duration (4-24 hours) to enhance proper folding and membrane insertion.

• Media supplementation: Enriched media formulations with appropriate osmolytes can improve membrane protein yields.

• Extraction protocols: Gentle detergent extraction using non-ionic detergents (DDM, LDAO) is typically required to solubilize membrane proteins while maintaining native structure.

For SG1469 specifically, the documented protocols have successfully employed E. coli expression systems with N-terminal 10xHis-tagging, enabling downstream purification by immobilized metal affinity chromatography (IMAC) .

How can lambda Red recombineering be applied to study SG1469 in Sodalis glossinidius?

Lambda Red recombineering represents a valuable genetic modification technique adapted for manipulating S. glossinidius, enabling precise genetic alterations to study genes like SG1469. The optimized methodology involves:

• Transformation with recombineering plasmid: Transform S. glossinidius with pKD46, which carries the arabinose-inducible lambda Red recombination genes .

• Growth conditions: Grow S. glossinidius harboring pKD46 with shaking to an OD600 of approximately 0.5 .

• Induction protocol: Induce cells in Mitsuhashi and Maramorosch (MM) medium supplemented with 0.5% (wt/vol) arabinose and 5 mM cAMP. Optimal induction time is approximately 0.5 hours to obtain hyper-recombinogenic S. glossinidius cells .

• Target sequence preparation: Prepare SG1469 replacement allele containing a selectable marker (e.g., antibiotic resistance gene) flanked by homologous sequences targeting SG1469. The length of homology regions significantly affects recombination efficiency, with longer flanking sequences yielding more recombinants .

• Transformation: Make cells chemically competent and transform with the prepared replacement allele (250 ng is sufficient) .

• Selection and verification: Select transformants on appropriate antibiotic-containing media and confirm successful recombination by PCR analysis using primers that flank the SG1469 gene .

This approach enables the generation of SG1469 knockout or modified strains in S. glossinidius, facilitating functional studies of this membrane protein in its native bacterial context. The technique is particularly valuable for investigating SG1469's role in symbiotic relationships with insect hosts .

What role might SG1469 play in heme tolerance in the tsetse fly gut environment?

While direct evidence specifically linking SG1469 to heme tolerance is not explicitly stated in the search results, the research context suggests potential involvement based on several findings:

• Heme-rich environment: S. glossinidius encounters high heme levels in the tsetse fly gut following blood meals, necessitating adaptive mechanisms to mitigate heme toxicity .

• Membrane protein function: As a multi-pass membrane protein, SG1469 may participate in transport processes or membrane integrity maintenance under heme stress conditions.

• Gene expression patterns: RNAseq analysis identified 436 genes differentially expressed in S. glossinidius under high heme conditions, including genes involved in inorganic ion transport and metabolism . The search results don't explicitly mention SG1469 among these genes, but as a membrane protein, it may play a role in related processes.

• Experimental approach: To investigate potential involvement of SG1469 in heme tolerance, researchers could:

  • Analyze SG1469 expression levels under varying heme concentrations

  • Generate SG1469 knockout mutants using lambda Red recombineering

  • Assess mutant colonization efficiency in tsetse fly guts following methodology described in search result :

    • Introduce wild-type or mutant S. glossinidius to heat-inactivated bovine blood at 500 CFU/ml

    • Provide to flies through an artificial membrane system

    • Maintain flies on heat-inactivated blood every 48 hours

    • Dissect gut tissues at 1, 5, and 10 days post-inoculation

    • Homogenize and plate gut tissues to quantify bacterial colonization levels

This experimental approach would determine whether SG1469 contributes to S. glossinidius colonization success in the heme-rich tsetse gut environment .

What experimental designs are most effective for studying SG1469 functions?

When designing experiments to investigate SG1469 functions, researchers should consider several experimental design approaches:

• Pre-test post-test control group design: This classic design allows for comparison between wild-type and SG1469-modified strains before and after experimental intervention :

  Group	Pre-test (O1)	Treatment (X)	Post-test (O2)
  Experimental	Measure	SG1469 modification	Measure
  Control	Measure	No modification	Measure

  This design controls for potential confounding variables and provides robust evidence of functional changes resulting from SG1469 modification .

• Removed-treatment design: This approach is particularly valuable for studying membrane protein functions:

  Observation (O1)	Treatment (X)	Observation (O2)	Remove Treatment	Observation (O3)
  Baseline measure	SG1469 expression	Functional measure	SG1469 inhibition	Recovery measure

  This design allows researchers to observe not only the effect of introducing functional SG1469 but also the consequences of its subsequent removal, providing stronger evidence for causality in observed phenotypes .

• Multiple-group comparative design: For comparing different SG1469 variants:

  Group	Pre-test	Treatment	Post-test
  Wild-type SG1469	O1	X1	O2
  Modified SG1469 variant 1	O3	X2	O4
  Modified SG1469 variant 2	O5	X3	O6

  This design enables researchers to compare functional differences between SG1469 variants with specific modifications .

The selection of optimal experimental design should be guided by the specific research question, available resources, and the biological context of SG1469 function under investigation.

How can researchers assess the functional integrity of recombinant SG1469?

Assessing the functional integrity of recombinant SG1469 requires a multi-faceted approach:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure integrity

  • Fluorescence spectroscopy to assess tertiary structure

  • Size-exclusion chromatography to confirm proper oligomeric state

• Membrane insertion validation:

  • Liposome reconstitution experiments

  • Proteoliposome flotation assays

  • Membrane fractionation of expression hosts followed by Western blotting

• Functional assays:

  • For transport functions: liposome-based transport assays with appropriate substrates

  • For structural roles: membrane integrity assays in the presence/absence of functional SG1469

  • In vivo complementation assays using SG1469 knockout strains

• Single-molecule tracking approaches:
  Based on methodologies in search result , researchers can assess membrane protein dynamics:

  • Fuse SG1469 with fluorescent proteins (e.g., GFP)

  • Employ fluorescence single-molecule tracking to measure diffusion characteristics

  • Compare behavior in different membrane environments (native vs. artificial)

  • Analyze diffusion coefficients and mobile fractions as indicators of proper membrane integration

• Structural integrity verification:

  • Limited proteolysis to confirm proper folding

  • Antibody binding assays targeting conformational epitopes

  • Mass spectrometry to verify post-translational modifications if present

These approaches collectively provide a comprehensive assessment of whether recombinant SG1469 maintains its native structural and functional characteristics, essential for valid experimental interpretations.

How can researchers modify SG1469 for functional studies without compromising its membrane characteristics?

Strategic modification of SG1469 for functional studies while preserving its membrane characteristics requires careful consideration of several factors:

• Tag position optimization:

  • N-terminal tags are generally preferred for multi-pass membrane proteins like SG1469

  • If C-terminal tagging is necessary, include flexible linker sequences to minimize interference with membrane domains

  • Consider removable tags with protease cleavage sites for post-purification tag removal

• Fusion protein design:

  • For fluorescent tagging, select monomeric fluorescent proteins that minimize oligomerization artifacts

  • Position fluorescent tags after careful bioinformatic analysis of predicted topology to avoid disrupting transmembrane domains

  • Consider split fluorescent protein approaches where complementary fragments are placed at different locations

• Site-directed mutagenesis strategies:

  • Target conserved residues identified through sequence alignment of UPF0060 family proteins

  • Employ conservative substitutions initially (e.g., Leu→Ile, Asp→Glu) to minimize structural disruption

  • Focus on charged residues in predicted loop regions as they often participate in functional interactions while being less critical for membrane integration

• Domain swapping approaches:

  • Identify discrete functional domains through bioinformatic analysis

  • Design chimeric constructs with homologous domains from related proteins

  • Maintain intact transmembrane segments to preserve membrane topology

• Conditional modification systems:

  • Implement chemically-induced dimerization systems for reversible functional alteration

  • Consider light-switchable domains for spatiotemporal control of protein function

  • Employ temperature-sensitive mutations for conditional function studies

Each modification strategy should be validated using the functional integrity assessments described in the previous section to ensure that the modified SG1469 protein maintains its native membrane characteristics while enabling the specific experimental manipulations required.

How does SG1469 compare to other membrane proteins in terms of diffusion dynamics?

While the search results don't provide direct diffusion data for SG1469 specifically, we can extrapolate from studies of other membrane proteins to establish expectations and experimental approaches:

• Membrane protein diffusion characteristics:
  Based on study , membrane proteins demonstrate distinct diffusion patterns depending on their structure and membrane interaction:

  Protein Type	Diffusion Coefficient Range	Mobile Fraction Characteristics
  GPI-anchored proteins	Relatively consistent across different polymer cushion lengths	Enhanced with increasing polymer length
  Single-pass transmembrane proteins	Increases with polymer cushion length	Enhanced with increasing polymer length
  Seven-pass transmembrane proteins	Increases with polymer cushion length	Enhanced with increasing polymer length

  As a multi-pass membrane protein, SG1469 would likely demonstrate diffusion behavior most similar to the seven-pass transmembrane proteins, with diffusion coefficients that increase when membrane-substrate distance is increased .

• Experimental approach for SG1469 diffusion studies:

  • Fluorescently label SG1469 (e.g., GFP fusion)

  • Prepare supported lipid bilayers (SLBs) with varying polymer cushion lengths

  • Employ single-molecule tracking to measure diffusion characteristics

  • Analyze transient confinement events and diffusion coefficients

  • Compare behavior with other membrane proteins of known structure

• Expected findings:

  • SG1469 likely exhibits slower diffusion compared to peripheral membrane proteins

  • Diffusion coefficient would likely increase with greater membrane-substrate separation

  • Mobile fraction would improve with longer polymer cushions

  • Size and multi-pass nature may result in diffusion coefficients lower than those of single-pass proteins

This comparative analysis provides a framework for designing and interpreting diffusion studies of SG1469, situating findings within the broader context of membrane protein dynamics .

What are the challenges and opportunities in using SG1469 for paratransgenic insect control strategies?

The potential use of SG1469 in paratransgenic insect control strategies presents both challenges and opportunities:

Opportunities:

• Endosymbiont engineering platform: S. glossinidius represents a valuable platform for paratransgenic approaches due to its stable association with tsetse flies . SG1469, as a membrane protein, could potentially serve as:

  • An anchoring system for surface display of anti-pathogen molecules

  • A secretion component for effector delivery

  • A target for conditional symbiont control

• Genetic manipulation tools: The optimized lambda Red recombineering strategy enables precise genetic modification of S. glossinidius and genes like SG1469, facilitating the development of engineered symbionts for paratransgenic applications .

• Heme adaptation mechanism: Understanding SG1469's potential role in heme tolerance could inform strategies for improving symbiont survival and persistence in the insect gut environment .

Challenges:

• Functional characterization: Limited knowledge of SG1469's specific function presents challenges for rational engineering. Additional research is needed to elucidate its precise role in S. glossinidius biology.

• Expression optimization: Ensuring stable expression of modified SG1469 variants in the symbiont context requires careful optimization of expression systems.

• In vivo validation: Testing engineered S. glossinidius strains with modified SG1469 requires complex in vivo experiments, as described in search result :

  • Introducing engineered bacteria to flies through artificial feeding systems

  • Monitoring colonization efficiency over time

  • Assessing impact on vector competence for pathogens

  • Evaluating long-term stability of genetic modifications

• Regulatory considerations: Deployment of paratransgenic strategies using engineered symbionts involves significant regulatory hurdles and ecological risk assessments.

What advanced imaging techniques are suitable for studying SG1469 localization and dynamics?

Advanced imaging techniques offer powerful approaches for investigating SG1469 localization and dynamics:

• Super-resolution microscopy:

  • Stimulated Emission Depletion (STED) microscopy: Achieves resolution below the diffraction limit, enabling visualization of SG1469 distribution within bacterial membranes

  • Photoactivated Localization Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM): Provides nanoscale resolution for precise localization studies

  • Structured Illumination Microscopy (SIM): Offers improved resolution for visualizing membrane protein organization

• Single-molecule tracking approaches:

  • Total Internal Reflection Fluorescence (TIRF) microscopy: Enables selective visualization of membrane-proximal SG1469 molecules with high signal-to-noise ratio

  • Single-particle tracking with photoactivatable fluorescent proteins: Allows for sparse labeling and tracking of individual SG1469 molecules

  • Fluorescence Recovery After Photobleaching (FRAP): Provides information on SG1469 mobility within membranes

• Correlative imaging approaches:

  • Correlative Light and Electron Microscopy (CLEM): Combines fluorescence localization with ultrastructural context

  • cryo-Electron Tomography: Enables visualization of SG1469 in the native membrane environment at molecular resolution

• Functional imaging techniques:

  • Förster Resonance Energy Transfer (FRET): Detects protein-protein interactions involving SG1469

  • Fluorescence Lifetime Imaging Microscopy (FLIM): Provides information on the microenvironment of labeled SG1469

  • Fluorescence Correlation Spectroscopy (FCS): Measures diffusion and concentration of SG1469 with high temporal resolution

• In vivo imaging approaches:

  • Fluorescent labeling of SG1469 in S. glossinidius within tsetse fly gut tissues

  • Multiphoton microscopy for deeper tissue imaging

  • Light-sheet microscopy for reduced phototoxicity in long-term imaging

These advanced imaging techniques, particularly when used in combination, can provide comprehensive insights into SG1469 localization, dynamics, and function in both reconstituted systems and the native bacterial context .

How can contradictory data in SG1469 research be reconciled through improved experimental design?

When researchers encounter contradictory data in SG1469 studies, several experimental design approaches can help reconcile discrepancies:

• Implement factorial experimental designs:

  • Systematically vary multiple experimental parameters simultaneously

  • Identify interaction effects that may explain contradictory outcomes

  • Example design for SG1469 expression studies:

  Temperature	Induction Duration	Expression Host	Membrane Fraction	Result
  18°C	4h	E. coli C41(DE3)	Measure	Data point 1
  18°C	4h	E. coli BL21(DE3)	Measure	Data point 2
  18°C	16h	E. coli C41(DE3)	Measure	Data point 3
  18°C	16h	E. coli BL21(DE3)	Measure	Data point 4
  30°C	4h	E. coli C41(DE3)	Measure	Data point 5
  etc.

• Apply Solomon four-group design:
  This robust design helps identify whether pre-testing or measurement approaches influence results :

  Group	Pre-test	Treatment	Post-test
  RA	O1	XA	O2
  RB	O3		O4
  RC		XB	O5
  RD			O6

  Where R represents randomization, O represents observations, and X represents experimental interventions. This design helps determine whether conflicting results arise from measurement artifacts or actual biological differences .

• Use multiple independent methods:

  • Apply different analytical techniques to the same biological question

  • Compare results from biochemical, biophysical, and genetic approaches

  • Triangulate findings to identify consistent patterns across methodologies

• Standardize experimental protocols:

  • Develop detailed standard operating procedures for SG1469 experiments

  • Control for variables like protein batch, buffer composition, and handling procedures

  • Implement blinded analysis where appropriate to reduce bias

By implementing these robust experimental design approaches, researchers can systematically identify sources of variation in SG1469 studies and reconcile apparently contradictory findings, advancing our understanding of this membrane protein's structure and function .