Recombinant UPF0317 protein SAV_6940 (SAV_6940)

What are the optimal expression systems for recombinant UPF0317 protein SAV_6940?

UPF0317 protein SAV_6940 can be expressed in multiple host systems, each offering distinct advantages. While Escherichia coli remains a common first choice for many recombinant proteins, alternative expression systems may yield superior results depending on your specific research requirements .

Comparative Expression Systems for UPF0317 protein SAV_6940:

Recent studies suggest that for some recombinant proteins, V. natriegens can overcome roadblocks encountered with E. coli expression, suggesting fundamental differences in processes such as protein folding via chaperones .

How can I optimize transformation and growth conditions for UPF0317 protein expression?

Successful expression begins with effective transformation and optimized growth conditions. For challenging proteins like UPF0317 protein SAV_6940, consider these evidence-based approaches:

For E. coli-based expression:

• Use BL21(DE3) or similar strains with reduced protease activity

• Optimize induction conditions: typically IPTG at 0.1-1.0 mM

• Consider lower temperature induction (16-20°C) to enhance protein folding

• Test auto-induction media for higher yields of some proteins

For V. natriegens-based expression:

• Use reduced ampicillin concentration (5 μg/mL initially, then 50 μg/mL for subsequent growth) to overcome higher sensitivity compared to E. coli

• Prepare higher density competent cells to improve transformation efficiency

• Modify transformation protocols by eliminating secondary heat shock steps and shortening grow-out times to 1 hour

• Consider maintaining cultures at 30°C rather than lower temperatures, as lower temperatures may reduce recombinant expression in V. natriegens

What purification strategies are most effective for UPF0317 protein SAV_6940?

Purification strategy depends on expression system and fusion tags incorporated into your construct. Based on experimental data with similar proteins, we recommend:

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

• Intermediate purification: Tag cleavage using TEV protease

• Polishing step: Size exclusion chromatography (SEC)

Key considerations:

• Expression in different hosts may affect purification outcomes; protein from V. natriegens has shown higher proportion of properly folded protein for some targets compared to E. coli

• Monitor for common contaminants - when expressed in E. coli, recombinant proteins may co-purify with ArnA (~75 kDa contaminant in IMAC)

• SEC profiles can indicate protein quality - properly folded proteins typically elute in the expected molecular weight range, while aggregates elute in the void volume

How does protein folding of UPF0317 protein SAV_6940 differ between expression systems?

Research suggests fundamental differences in protein folding between expression systems, with significant implications for research outcomes:

Case study evidence from similar proteins:

• Small GTPase KRAS4b expressed in V. natriegens showed almost complete digestion by TEV protease and monomeric behavior on SEC, indicating proper folding

• The same protein expressed in E. coli exhibited partial resistance to TEV protease and contained soluble aggregates, suggesting incomplete folding

• RAF1 kinase CR1 domain showed improved thermal stability (measurable Tm of 72-74°C) when expressed in V. natriegens versus unmeasurable Tm for E. coli-expressed protein

This suggests that while E. coli may produce higher total amounts of soluble fusion protein, V. natriegens may produce a higher proportion of correctly folded protein leading to better final yields of functional protein .

Hypothesized mechanisms for these differences include:

• Differences in chaperone availability or activity

• Variation in cytoplasmic redox environment

• Differences in translation rates affecting co-translational folding

What strategies can improve isotopic labeling of UPF0317 protein SAV_6940 for structural studies?

Isotopic labeling is essential for NMR studies and can enhance mass spectrometry analyses. Based on experimental findings with other recombinant proteins:

Optimization strategies for isotopic labeling:

For UPF0317 protein SAV_6940, V. natriegens expression may be particularly valuable if the protein proves challenging to express in E. coli, as it can provide sufficient material for structural biology applications that would otherwise be unaffordable or impossible to produce .

How can analytical techniques be used to assess the quality of recombinant UPF0317 protein SAV_6940?

Multiple complementary techniques should be employed to fully characterize recombinant UPF0317 protein:

Recommended analytical cascade:

• SDS-PAGE/Coomassie staining: Initial purity assessment (target >95% purity)

• Size Exclusion Chromatography (SEC): Evaluate oligomeric state and aggregation

• Mass Spectrometry:

  • Intact mass analysis: Verify protein identity and post-translational modifications

  • Peptide mapping: Confirm sequence coverage and identify modifications

• Thermal stability assessment:

  • Differential Scanning Fluorimetry (DSF): Determine melting temperature (Tm)

  • Note: Poorly folded proteins may show high initial background fluorescence in DSF due to exposed hydrophobic regions

• Functional assays:

  • Activity tests specific to the protein class

  • Binding assays if interaction partners are known

When comparing expression systems, these analytical techniques can reveal important differences in protein quality. For example, the SEC profile can indicate if the protein elutes as expected for its molecular weight or shows signs of aggregation or interaction with the column matrix, as observed with some nanobodies expressed in different systems .

What are the potential functions of UPF0317 protein SAV_6940 based on structural and sequence analyses?

While specific functional data for UPF0317 protein SAV_6940 is limited, bioinformatic approaches can provide insights:

• Sequence homology: Analysis of conserved domains may reveal relationships to characterized protein families

• Structural prediction: Tools like AlphaFold can predict 3D structure to suggest functional sites

• Genomic context: Examining neighboring genes may indicate involvement in specific pathways

• Comparative expression analyses: Transcriptomic data might reveal co-expression patterns suggesting functional relationships

A differentially expressed gene analysis mentioned in search results identified UPF0317 protein SAV_6940 (comp103200_c0_seq1) with a fold change of 4.88 (p=0.03) , suggesting potential biological significance under specific conditions.

How should I design expression constructs for optimal UPF0317 protein SAV_6940 production?

Construct design significantly impacts expression success. Consider these evidence-based approaches:

Key design elements:

• Vector selection: pET-series vectors work well in both E. coli and V. natriegens

• Fusion tags:

  • N-terminal His6 tag facilitates purification

  • MBP (maltose-binding protein) can enhance solubility

  • Consider TEV protease cleavage sites for tag removal

• Codon optimization: Adapt to the expression host of choice

• Regulatory elements: Strong promoters (T7) for high expression, weaker promoters if toxicity is observed

Construct evaluation strategies:

• Test multiple constructs in parallel small-scale expressions

• Compare different fusion partners' effects on solubility

• Evaluate the impact of N- versus C-terminal tags

For expression in V. natriegens, constructs developed for E. coli can often be used directly, but consider ampicillin resistance issues noted earlier .

What are the critical parameters for scaling up UPF0317 protein SAV_6940 production for structural biology applications?

Scaling from small test expressions to larger preparations requires careful optimization:

Critical parameters for scale-up:

The faster growth rate of V. natriegens (≤10 min doubling time) allows generation of cell pellets within 24 hours from seed culture inoculation, substantially improving laboratory throughput compared to typical E. coli processes .

How can I troubleshoot common problems with UPF0317 protein SAV_6940 expression and purification?

Based on experience with similar proteins, here are solutions to common challenges:

Troubleshooting guide:

When encountering difficulty with expression in E. coli, consider V. natriegens as an alternative, particularly for proteins that form inclusion bodies or show poor folding .

What emerging technologies are enhancing recombinant protein expression for proteins like UPF0317 SAV_6940?

Recent advances are expanding options for challenging protein expression:

• Cell-free protein synthesis systems:

  • Eliminate cell viability constraints

  • Rapid production (hours versus days)

  • Easily incorporate non-canonical amino acids

• Engineered expression hosts:

  • V. natriegens with expanded genetic tools

  • E. coli strains with enhanced disulfide bond formation

  • Yeast strains with humanized glycosylation pathways

• AI-assisted protein design:

  • Improved prediction of solubility-enhancing mutations

  • Optimization of constructs for specific expression systems

• High-throughput screening platforms:

  • Parallel testing of multiple expression constructs

  • Automated purification and analysis

These technologies are particularly valuable for structurally complex or poorly characterized proteins like UPF0317 SAV_6940, where traditional approaches may yield insufficient material for advanced structural and functional studies.

How does the research on UPF0317 protein SAV_6940 contribute to broader scientific understanding?

While specific functions of UPF0317 protein SAV_6940 remain to be fully characterized, research on this protein contributes to several important scientific areas:

• Methodological advances in protein production:

  • Comparison between E. coli and V. natriegens expression systems provides insights into protein folding mechanisms

  • Optimization strategies developed may benefit production of other challenging proteins

• Bacterial physiology:

  • Understanding UPF0317 protein SAV_6940 may reveal aspects of bacterial adaptation mechanisms

  • Comparative studies between different bacterial species expressing homologous proteins can illuminate evolutionary relationships

• Structural biology:

  • Novel protein structures expand our understanding of protein folding and function

  • May reveal previously uncharacterized protein folds or functional motifs

• Potential biotechnological applications:

  • If functional characterization reveals enzymatic activity, potential applications in biocatalysis

  • Understanding protein folding differences between expression systems has broad implications for recombinant protein production technologies

This research exemplifies how studies of individual proteins contribute to both fundamental understanding of biological systems and practical advances in biotechnology methodologies.