Recombinant Beijerinckia indica subsp. indica Membrane protein insertase YidC (yidC)
Description
Introduction to Membrane Protein Insertase YidC
YidC represents a prominent member of the Oxa1 superfamily of membrane protein insertases that are widely distributed across bacterial species, including Beijerinckia indica subsp. indica. This essential protein plays a fundamental role in the biogenesis of bacterial inner membranes, significantly influencing both membrane protein composition and lipid organization . The YidC protein facilitates the proper integration and folding of numerous membrane proteins, making it indispensable for cellular viability and function in most bacterial species.
The evolutionary conservation of YidC across diverse bacterial phyla underscores its biological importance. In bacterial systems, YidC operates through two primary mechanisms: it collaborates with the Sec translocon to aid in the folding of multi-pass membrane proteins, and it functions independently as both an insertase and lipid scramblase to facilitate the integration of smaller membrane proteins while contributing to bilayer organization . This functional versatility highlights YidC's central role in maintaining membrane integrity and functionality.
The recombinant production of Beijerinckia indica subsp. indica YidC represents an important advancement in studying this specific homolog, potentially revealing species-specific adaptations in membrane protein biogenesis. While most mechanistic insights have been derived from studies in model organisms like Escherichia coli, the knowledge gained provides a valuable framework for understanding YidC function in diverse bacterial species, including Beijerinckia indica.
Biochemical Functions of YidC
The YidC protein performs several critical biochemical functions that contribute to bacterial membrane biogenesis. Primarily, it serves as an insertase that facilitates the integration of membrane proteins into the lipid bilayer. This function is particularly important for smaller membrane proteins, including phage coat proteins like M13 procoat and Pf3, as well as cellular proteins such as ATP synthase subunit c (F0c) and SecG .
Beyond its insertase activity, YidC also functions as a lipid scramblase, contributing to the organization and distribution of lipids between membrane leaflets . This dual functionality highlights YidC's comprehensive role in maintaining membrane integrity while simultaneously assisting in the proper arrangement and insertion of membrane proteins.
In collaborative contexts, YidC works with the Sec translocon to ensure proper folding of multi-pass membrane proteins. This cooperation represents a sophisticated mechanism for handling complex membrane proteins that require coordinated insertion and folding processes. Independently, YidC can facilitate the insertion of smaller membrane proteins without requiring additional protein machinery .
Recent experimental evidence indicates that YidC substrates, including M13 procoat, Pf3 coat proteins, and F0c, show significantly enhanced insertion efficiency when YidC functions in concert with its interacting partner YibN . This finding suggests that YidC's activity is regulated by accessory proteins that modulate its function in response to cellular needs.
YidC in Beijerinckia indica subsp. indica
The Beijerinckia indica subsp. indica YidC likely shares fundamental functional characteristics with its homologs in other bacterial species while potentially exhibiting unique adaptations suited to this organism's specific membrane environment. As a nitrogen-fixing bacterium with distinctive physiological properties, Beijerinckia indica may have evolved specialized features in its membrane protein biogenesis machinery.
The genomic context of YidC in Beijerinckia indica could provide insights into its regulation and functional partners specific to this organism. Comparative genomic analyses across bacterial species typically reveal both conserved elements and species-specific adaptations in membrane protein biogenesis pathways.
While direct experimental data on Beijerinckia indica YidC remains limited, comparative analysis with well-characterized homologs provides a foundation for understanding its likely structure and function. The recombinant production of this specific YidC variant offers opportunities to explore its biochemical properties and functional behaviors in controlled experimental settings.
Production of Recombinant Beijerinckia indica YidC
Recombinant production of membrane proteins like YidC presents significant technical challenges due to their hydrophobic nature and complex folding requirements. Expressing Beijerinckia indica subsp. indica YidC in heterologous systems typically requires careful optimization of expression conditions to ensure proper folding and functionality.
Common expression systems for recombinant membrane proteins include modified E. coli strains, yeast systems, or insect cell lines. For YidC specifically, E. coli-based expression systems have been successfully used for homologous proteins, incorporating affinity tags such as polyhistidine tags to facilitate purification . Similar approaches likely apply to the recombinant production of Beijerinckia indica YidC.
Purification typically involves membrane solubilization using detergents like DDM (n-dodecyl β-D-maltoside), followed by affinity chromatography and additional purification steps. The choice of detergent is crucial for maintaining protein stability and function throughout the purification process. For functional studies, the purified protein may be reconstituted into lipid bilayers or proteoliposomes to assess its native activities .
One significant challenge in producing recombinant YidC is maintaining its structural integrity and function outside its native membrane environment. Careful selection of detergents and buffer conditions is essential for preserving the protein's functional properties during purification and subsequent experimental applications.
Interaction Partners of YidC
Recent research has identified YibN as a significant physical and functional interactor of YidC in E. coli, with potential implications for understanding similar interactions in Beijerinckia indica YidC . This interaction was discovered through proximity-dependent biotin labeling (BioID) and subsequently validated using affinity purification-mass spectrometry assays and on-gel binding assays with purified proteins .
The YidC-YibN interaction depends critically on the transmembrane segment of YibN, suggesting that their association occurs within the hydrophobic interior of the lipid bilayer . This finding highlights the importance of membrane-embedded domains in mediating functional protein-protein interactions within the bacterial membrane.
Functionally, YibN enhances the production and membrane insertion of YidC substrates, including phage coat proteins and various small membrane proteins like SecG . This cooperative action significantly amplifies YidC's insertase capability, suggesting that YibN serves as an important regulator or facilitator of YidC-mediated membrane protein biogenesis.
Additional interaction partners for YidC include the protease FtsH and its regulatory partners, HflC and HflK, emphasizing connections between membrane protein insertion and quality control mechanisms . These interactions form part of a complex network that coordinates membrane protein biogenesis and maintenance in bacterial cells.
Functional Experiments and Research Findings
Experimental approaches to studying YidC function have yielded significant insights that likely apply to Beijerinckia indica YidC as well. In vitro translation and insertion assays using inverted membrane vesicles (INVs) have demonstrated that membranes enriched with YibN show enhanced insertion of YidC substrates, including Pf3 coat, M13 procoat, F0c, and SecG .
Co-expression studies have similarly revealed that YibN significantly enhances the production of various YidC substrates in vivo . This effect appears to be specific to YidC-dependent membrane proteins, as proteins like YajC and YhcB, whose insertion is not affected by YidC depletion, showed no enhancement when co-expressed with YibN .
Interestingly, YibN overexpression leads to membrane lipid production alteration and inner membrane proliferation, suggesting that it may interfere with YidC's lipid transport activity . This observation connects protein insertion functions with membrane lipid organization, highlighting the multifaceted role of these proteins in membrane biogenesis.
The specificity of YibN's effect appears to depend on the hydrophobicity of transmembrane segments. For instance, a SecG variant carrying the I20E mutation in its first transmembrane segment showed reduced enhancement by YibN compared to wild-type SecG . This finding suggests that the hydrophobic properties of substrate proteins influence their processing by the YidC-YibN system.
Future Research Directions
Future research on Beijerinckia indica subsp. indica YidC should focus on characterizing species-specific features that might distinguish it from homologs in other bacteria. Comparative functional studies between recombinant Beijerinckia indica YidC and well-characterized homologs could reveal adaptations specific to this nitrogen-fixing bacterium.
Structural studies using techniques like cryo-electron microscopy could provide valuable insights into the three-dimensional architecture of Beijerinckia indica YidC and its interactions with partner proteins. Such structural information would enhance our understanding of the molecular mechanisms underlying its function.
Investigation of Beijerinckia indica YidC's substrate specificity and interactome would further illuminate its role in this specific organism's membrane protein biogenesis. Identification of species-specific substrates or regulatory partners could reveal unique aspects of membrane biology in this bacterium.
The potential biotechnological applications of recombinant Beijerinckia indica YidC also merit exploration. Given YidC's role in membrane protein insertion, engineered variants could potentially serve as tools for heterologous membrane protein production or as targets for novel antimicrobial compounds.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it when placing your order. We will prepare the product accordingly.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional charges will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: bid:Bind_3328
STRING: 395963.Bind_3328
Q&A
What is the fundamental role of YidC in bacterial cells?
YidC functions as a membrane protein insertase that facilitates the insertion of newly synthesized proteins into lipid membranes. It serves a dual role in bacterial systems, acting either independently as a membrane insertase or as a chaperone in the SecYEG complex mechanism . YidC is required for the insertion, proper folding, and complex formation of integral membrane proteins. It's involved in integrating membrane proteins that insert both dependently and independently of the Sec translocase complex, and aids in the folding of multispanning membrane proteins .
How does the structure of YidC contribute to its function as an insertase?
The YidC protein contains a hydrophilic groove inside the membrane core that is critical for accepting the hydrophilic moieties of substrate proteins into the membrane. The hydrophilic region traverses the inner side of the membrane and is closed to the periplasmic side of the bilayer, which decreases the hydrophobicity of the membrane towards the external side of the lipid bilayer . This structural arrangement facilitates a two-step mechanism: first, YidC interacts with a hydrophilic protein region temporarily in its groove, and second, this peptide is translocated to the periplasmic side .
What are the key conserved domains in Beijerinckia indica YidC compared to other bacterial homologs?
While specific information about Beijerinckia indica YidC domains is limited in the available literature, generally YidC proteins share a conserved structural core composed of a membrane-embedded H1/4/5 helix bundle and a peripheral H0 brace . YidC contains a hairpin-interrupted three-TMH (transmembrane helix) motif that is structurally similar across bacterial species . The conservation of these structural elements suggests functional importance across bacterial species, including Beijerinckia indica.
What expression systems are optimal for producing functional recombinant Beijerinckia indica YidC?
For expressing recombinant Beijerinckia indica YidC, E. coli expression systems are commonly employed. When designing expression constructs, consider including an affinity tag (such as a His-tag) to facilitate purification . For optimal expression, the following methodological approach is recommended:
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Clone the full-length YidC sequence (604 amino acids for Beijerinckia indica YidC) into an appropriate expression vector
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Transform into an E. coli expression strain optimized for membrane proteins
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Culture in rich media (such as TB or 2xYT) supplemented with appropriate antibiotics
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Induce protein expression at lower temperatures (16-25°C) to facilitate proper membrane insertion
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Extract using mild detergents suitable for membrane proteins (DDM, LMNG, or Triton X-100)
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Purify using affinity chromatography followed by size exclusion chromatography
How can researchers effectively assess the functional activity of recombinant YidC?
Assessing YidC functionality requires measuring its ability to facilitate membrane protein insertion. A methodological approach involves:
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In vitro reconstitution assays: Incorporate purified YidC into liposomes and measure insertion of model substrates like Pf3 coat protein
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Complementation tests: Express recombinant YidC in YidC-depleted bacterial strains and assess restoration of growth
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Single-molecule force spectroscopy: Monitor how YidC guides the folding of substrate proteins into membranes in real-time
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Cross-linking experiments: Use techniques like pBpa (p-benzoyl-L-phenylalanine) incorporation or DSS (disuccinimidyl suberate) cross-linking to identify YidC interactions with substrate proteins
What storage conditions maximize stability and activity of purified recombinant YidC?
To maintain optimal stability and activity of purified recombinant YidC:
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Store in Tris/PBS-based buffer, pH 8.0, with 50% glycerol at -20°C/-80°C for long-term storage
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For working solutions, maintain 5-50% glycerol and store at 4°C for up to one week
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Avoid repeated freeze-thaw cycles, which can denature the protein
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Consider lyophilization in buffer containing 6% trehalose for extended shelf life (up to 12 months)
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Upon reconstitution, use a final protein concentration of 0.1-1.0 mg/mL
How does YidC facilitate the insertion of substrates with varying hydrophobicity profiles?
YidC employs a sophisticated mechanism that accommodates substrates with different hydrophobicity profiles. The hydrophilic groove within YidC's transmembrane domain serves as a crucial recognition and processing site for substrate proteins . During insertion, YidC undergoes conformational changes, and the hydration and dehydration of its hydrophilic groove are critical for substrate accommodation. The mechanism varies based on:
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Substrate recognition: YidC initially interacts with hydrophilic portions of the substrate protein within its groove
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Conformational adaptation: As observed with Pf3 coat protein, the substrate undergoes conformational changes (measured by bending angle analysis) to adapt to the YidC groove environment
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Transmembrane contact points: Substrates contact different YidC residues across four of its six transmembrane regions located in the inner leaflet, center, and periplasmic leaflet, providing a hydrophobic platform for insertion
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Translocation mechanism: YidC facilitates translocation by decreasing local membrane hydrophobicity and creating a favorable energetic environment for substrate passage
What is the molecular basis for YidC's dual function as both an independent insertase and a SecYEG-associated chaperone?
YidC's dual functionality stems from its versatile structure and interaction capabilities:
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Independent insertase mode: YidC can function autonomously for certain substrates, like small phage coat proteins Pf3 and M13, by providing a hydrophobic surface for peptide insertion into the lipid bilayer
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SecYEG-associated chaperone mode: YidC assists in the proper folding of proteins during SecYEG-mediated insertion, as demonstrated with the LacY lactose permease membrane protein
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Conformational flexibility: YidC undergoes significant conformational changes (RMSD >4 Å) when interacting with substrates, allowing it to adapt to different insertion scenarios
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Interaction network: YidC forms specific contacts with both the SecYEG translocon and SRP-targeting machinery, enabling coordinated protein insertion
This dual function is demonstrated in the case of the melibiose permease MelB, which can insert via YidC in the absence of SecYEG, with YidC chaperoning the stepwise insertion and folding process of both MelB folding cores .
How do single amino acid substitutions in the transmembrane domains of YidC affect its insertase activity?
Single amino acid substitutions in YidC's transmembrane domains can significantly impact its insertase function. Evidence from cysteine scanning and disulfide formation studies reveals:
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Critical contact points: Substrate proteins like Pf3 coat protein form contacts with specific YidC residues across four transmembrane regions
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Functional importance: When multiple contacting residues are mutated to serines, YidC function is severely disrupted, failing complementation tests
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Leaflet-specific interactions: Contact residues are distributed throughout the membrane bilayer (inner leaflet, center, and periplasmic leaflet), indicating a comprehensive interaction surface
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Insertion defects: Pf3 mutants with membrane insertion defects specifically fail to contact periplasmic residues of YidC, highlighting the importance of these interactions
These findings suggest that the spatial arrangement of specific amino acids throughout YidC's transmembrane domains creates an insertion pathway for substrate proteins.
What evolutionary relationship exists between YidC and the SecY translocon complex?
The evolutionary relationship between YidC and SecY reveals surprising connections:
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Shared structural core: SecY and YidC share a structural core composed of a membrane-embedded H1/4/5 bundle and a peripheral H0 brace, suggesting homology
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Evolutionary model: Evidence suggests that SecY might have originated from a YidC homolog that formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer
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Conserved dimerization: YidC preserves a tendency to form dimers via the same interface as the proposed SecY progenitor, with novel heterodimers formed by archaeal and eukaryotic YidC variants
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Functional conservation: Both proteins facilitate membrane protein insertion, with partially overlapping but distinct substrate preferences
This evolutionary relationship suggests that the SecY-dependent and YidC-dependent membrane insertion pathways evolved from a common ancestral mechanism.
What strategies can overcome solubility and stability issues when working with recombinant YidC?
Membrane proteins like YidC present unique challenges for recombinant expression and purification. Methodological approaches to overcome these challenges include:
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Optimized extraction: Use mild detergents like DDM, LMNG, or CHAPS that maintain the native structure of membrane proteins
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Stabilizing additives: Include glycerol (5-50%) in all buffers to enhance stability
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Lipid supplementation: Add phospholipids (0.1-0.5 mg/mL) to purification buffers to stabilize the native conformation
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Temperature management: Perform all purification steps at 4°C to minimize protein degradation
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Buffer optimization: Use Tris/PBS-based buffers at pH 8.0 with appropriate salt concentration (typically 150-300 mM NaCl)
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Construct engineering: Consider expressing functional fragments or creating fusion constructs with stabilizing partners
How can researchers distinguish between SecYEG-dependent and YidC-independent insertion pathways in experimental systems?
Distinguishing between insertion pathways requires carefully designed experimental approaches:
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Conditional depletion systems: Use E. coli strains with conditionally depleted SecYEG or YidC to assess substrate insertion in the absence of either pathway
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In vitro reconstitution: Prepare proteoliposomes containing either purified YidC, SecYEG, or both, and compare insertion efficiency of model substrates
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Cross-linking analysis: Perform site-specific cross-linking to identify interaction partners during insertion
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Co-expression systems: Establish co-expression of YidC and SecYEG at controlled ratios to analyze complex formation and substrate preferences
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Single-molecule approaches: Use single-molecule force spectroscopy to monitor real-time insertion and folding processes mediated by different insertion machinery
This methodological approach revealed that membrane proteins like MelB can insert via the YidC pathway in the absence of SecYEG, demonstrating pathway-specific capabilities .
What molecular dynamics simulation approaches best capture YidC-mediated insertion mechanisms?
Molecular dynamics (MD) simulations offer powerful tools for investigating YidC-mediated insertion at the atomic level. Methodological considerations include:
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Simulation types: Combine equilibrium and non-equilibrium MD simulations to fully capture the dynamic insertion process
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Docking models: Build multiple docking models of YidC with substrate proteins (e.g., YidC-Pf3) to explore different conformational states during insertion
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Analysis metrics: Monitor parameters like RMSD, bending angles, and hydration states to characterize conformational changes during insertion
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Membrane environment: Use appropriate lipid bilayer compositions that mimic bacterial membranes
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Simulation timescales: Extend simulations to microsecond timescales to observe complete insertion events
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Enhanced sampling: Apply techniques like umbrella sampling or metadynamics to overcome energy barriers and explore rare events in the insertion process
MD simulations have revealed that during insertion, both YidC and substrate proteins undergo significant conformational changes, with hydration and dehydration of YidC's hydrophilic groove playing critical roles .
How might high-resolution structural studies of Beijerinckia indica YidC advance our understanding of insertase mechanisms?
High-resolution structural studies of Beijerinckia indica YidC would advance the field by:
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Revealing species-specific adaptations in the membrane insertion machinery
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Identifying unique structural features that might confer substrate specificity
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Providing comparative models to understand YidC evolution across bacterial lineages
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Informing the design of specific inhibitors or modulators of bacterial membrane protein insertion
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Elucidating the atomic basis for YidC's dual function as both an independent insertase and SecYEG-associated chaperone
What potential biotechnological applications might emerge from detailed understanding of YidC-mediated insertion?
Advanced understanding of YidC-mediated insertion mechanisms opens several biotechnological avenues:
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Engineered membrane protein expression systems: Optimized co-expression of YidC variants could enhance production of challenging membrane proteins for structural studies
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Antimicrobial development: YidC-specific inhibitors could represent a novel class of antibiotics targeting essential membrane protein biogenesis
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Synthetic biology tools: Engineered YidC variants could facilitate insertion of non-natural membrane proteins into artificial cell systems
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Protein engineering: Understanding insertion mechanics could guide the design of membrane proteins with enhanced stability or function
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Nanobiotechnology: YidC-mediated insertion could be harnessed for preparing protein-functionalized liposomes and nanoparticles for drug delivery
