Recombinant UPF0603 protein Rv2345/MT2410 (Rv2345, MT2410)

What is UPF0603 protein Rv2345/MT2410 and what are its basic structural features?

UPF0603 protein Rv2345/MT2410 is a conserved membrane protein encoded by the Rv2345 gene in the Mycobacterium tuberculosis H37Rv genome. The protein has the following characteristics:

• Gene location: 2623821 - 2625803 (+) in the MTB genome

• Protein size: 661 amino acids (full length)

• Gene length: 1983 base pairs

• UniProt accession number: P95241

• Expression region: 27-660 amino acids

The protein contains transmembrane domains as evidenced by its amino acid sequence, which includes hydrophobic regions characteristic of membrane proteins. According to sequence analysis, it contains the segment "RVVLLVTVGIIVIVVAVLLVVMRHRNRRRR" which strongly suggests a transmembrane region .

What expression systems have been successfully used for Rv2345/MT2410 production?

Based on available research data, several expression systems have been employed for recombinant production of mycobacterial proteins like Rv2345/MT2410:

For membrane proteins like Rv2345/MT2410, E. coli systems often require careful optimization of induction conditions. Research indicates that using lower temperatures (25°C vs. 37°C) and reduced IPTG concentrations (0.1 mM) can significantly improve soluble protein expression .

What storage conditions should be used for maintaining recombinant Rv2345/MT2410 stability?

According to product literature and research protocols, the following storage conditions are recommended :

• Short-term storage: Working aliquots can be stored at 4°C for up to one week

• Medium-term storage: -20°C in a Tris-based buffer with 50% glycerol

• Long-term storage: -80°C in small aliquots to prevent freeze-thaw cycles

• Important note: Repeated freezing and thawing is not recommended as it may compromise protein structure and activity

The protein is typically stored in a Tris-based buffer optimized specifically for this protein, containing 50% glycerol as a cryoprotectant to maintain stability during freeze-thaw cycles .

What experimental design approaches should be considered for optimizing Rv2345/MT2410 expression?

A multivariate Design of Experiments (DoE) approach is strongly recommended over traditional one-factor-at-a-time methods for optimizing Rv2345/MT2410 expression. Research demonstrates that this approach:

• Allows simultaneous evaluation of multiple variables affecting protein expression

• Identifies significant interactions between variables that would be missed in univariate approaches

• Reduces the total number of experiments needed

• Provides robust statistical validation of results

For membrane proteins like Rv2345/MT2410, a 2^8-4 fractional factorial design has been successfully employed to optimize expression conditions by evaluating variables such as:

• Induction absorbance (cell density at induction)

• IPTG concentration

• Expression temperature

• Media composition components (yeast extract, tryptone, glucose, glycerol)

• Antibiotic concentration

Statistical analysis from similar experimental designs has shown that for membrane proteins, the following factors are often significant (p<0.1) for maximizing soluble protein expression:

• Induction at mid-exponential phase (OD600 of 0.8)

• Lower induction temperature (25°C)

• Moderate levels of yeast extract and tryptone (5 g/L each)

• Lower IPTG concentration (0.1 mM)

How can the Clp proteolytic machinery influence recombinant expression of proteins in Mycobacterium systems?

The Clp proteolytic machinery plays a crucial role in Mycobacterium tuberculosis protein homeostasis and can significantly impact recombinant protein expression. Recent research has established that:

• The Clp machinery in MTB consists of:

  • ClpP1P2 protease core (a barrel-shaped heterotetradecameric complex)

  • Hexameric ring-like ATP-dependent unfoldases (ClpX or ClpC1)

• Both ClpC1 and ClpP1P2 are essential for:

  • Extracellular growth of MTB

  • Survival in macrophages

  • Maintenance of protein homeostasis

• The proteolytic system regulates dosage-sensitive proteins, especially those with:

  • Intrinsically disordered regions at their termini

  • Complex quaternary structures (like dodecameric conformations)

For recombinant expression of MTB proteins, researchers should consider:

• Co-expression systems that account for the natural proteolytic regulation in the native organism

• Potential degradation patterns if expressing in homologous systems

• Protein stabilization strategies when the recombinant protein might be a substrate for Clp machinery

Research has identified that approximately 38% of proteins regulated by the Clp machinery are essential for in vitro growth of MTB, highlighting the importance of this system in protein regulation .

What purification strategies are most effective for membrane proteins like Rv2345/MT2410?

Purifying membrane proteins like Rv2345/MT2410 presents unique challenges compared to soluble proteins. Based on research literature, the following approaches have proven effective:

• Affinity tag selection and placement:

  • Placing tags at the N-terminus rather than C-terminus often improves accessibility

  • His-tags remain the most versatile for membrane proteins

  • Consider using intein-based systems for tag removal without proteases

• Detergent selection for membrane protein extraction:

  • A systematic screening of detergents is essential

  • Mild non-ionic detergents (DDM, LMNG) often preserve structure

  • Detergent concentration must be optimized to prevent aggregation

• Purification protocol optimization:

  • Two-step purification is typically required (affinity followed by size exclusion)

  • All buffers should contain detergent above critical micelle concentration

  • Consider using lipid nanodiscs for native-like environment

For Rv2345/MT2410 specifically, research suggests maintaining the protein in a 50% glycerol buffer with appropriate detergents throughout the purification process to maintain stability and prevent aggregation .

What approaches can improve solubility of membrane proteins like Rv2345/MT2410 during recombinant expression?

Improving the solubility of membrane proteins remains one of the most challenging aspects of recombinant protein expression. For proteins like Rv2345/MT2410, several research-validated strategies have shown promise:

• Expression condition optimization using DoE approaches:
  Statistical analysis from fractional factorial designs shows that the following conditions significantly impact solubility:

  Variable	Optimal condition	Effect on solubility	p-value
  Induction temperature	25°C	Positive	<0.001
  IPTG concentration	0.1 mM	Positive	0.038
  Yeast extract	5 g/L	Positive	<0.001
  Tryptone	5 g/L	Positive	0.0027
  Glucose	1 g/L	Positive	0.0685

• Fusion protein approaches:

  • N-terminal fusions with highly soluble partners (MBP, SUMO, Trx)

  • C-terminal stability enhancers

  • Intein-based systems that allow precise control of protein cleavage

• Co-expression with chaperones:

  • GroEL/GroES system

  • DnaK/DnaJ/GrpE combinations

  • Specific lipid environment reconstitution

• Directed evolution of expression constructs:

  • Systematic truncation of hydrophobic regions

  • Surface engineering to increase polar interactions

  • Fusion with solubility enhancing peptides

For Rv2345/MT2410 specifically, examining the amino acid sequence reveals transmembrane segments that challenge soluble expression. Research indicates that engineering constructs that express only the soluble domains while preserving functional epitopes may be a viable approach .

What functional assays can be used to assess the activity of recombinant Rv2345/MT2410?

Despite being classified as a protein with unknown function (UPF), several approaches can be employed to assess the functional characteristics of Rv2345/MT2410:

• Structural characterization:

  • Circular dichroism to confirm proper folding

  • Limited proteolysis to identify stable domains

  • Thermal shift assays to assess stability

• Interaction studies:

  • Pull-down assays with MTB lysate to identify binding partners

  • Bacterial two-hybrid systems

  • Crosslinking followed by mass spectrometry

  • Analysis of protein-protein interactions using the String database

• Localization and membrane integration:

  • Fluorescent protein fusions to confirm cellular localization

  • Protease protection assays to determine topology

  • Reconstitution in liposomes to assess membrane integration

• Comparative analysis with orthologs:
  According to the TB database, Rv2345 belongs to orthogroup 882, with related genes in several mycobacterial species:

  • CE2175, cg2496, DIP1705, jk0617, MAP2133, MAV_2041

  • Mkms_3507, Mmcs_3444, MSMEG_4484, MT2410

  • Mvan_3816, nfa14640, SAV3184

  Functional information from these orthologs may provide insights into Rv2345 function.

• Phenotypic assays:

  • Complementation studies in knockout strains

  • Overexpression phenotype analysis

  • Growth studies under various stress conditions

Due to the essential nature of many MTB proteins involved in membrane processes and cell wall biosynthesis, correlation with phenotypic data from ClpC1 or ClpP2 knockdown strains may provide functional clues, as the Clp machinery has been shown to regulate many essential proteins in MTB .

How can researchers address the challenges of post-translational modifications when expressing Rv2345/MT2410 in heterologous systems?

Expressing mycobacterial membrane proteins in heterologous systems presents challenges related to post-translational modifications. Research indicates several approaches to address these issues:

• Selection of appropriate expression system based on modification requirements:

  Expression System	Modification Capability	Suitability for Rv2345/MT2410
  E. coli	Limited PTMs, no glycosylation	Good for basic structure studies
  Mycobacterium smegmatis	Similar PTM machinery to MTB	Excellent for native-like modifications
  Brevibacillus	Efficient secretion, some PTMs	Good for secreted domains
  Mammalian cells	Complex PTMs, glycosylation	Best for interaction studies

• Modification-specific approaches:

  • Phosphorylation sites can be mimicked by glutamate substitutions

  • Glycosylation sites can be identified and mutated if not essential

  • Lipidation motifs can be preserved or modified depending on experimental goals

• Co-expression with modification enzymes:

  • MTB-specific chaperones

  • Mycobacterial-specific modification enzymes

  • Specialized folding assistants

• Hybrid approaches:

  • Expression of difficult domains separately

  • Chemical ligation of separately expressed domains

  • Synthetic biology approaches combining chemical and biological synthesis

For Rv2345/MT2410 specifically, it's important to consider its native membrane environment in MTB. The protein sequence "RVVLLVTVGIIVIVVAVLLVVMRHRNRRRR" suggests a transmembrane domain followed by a positively charged region, which may interact with mycobacterial-specific lipids . These interactions may be critical for proper folding and function, necessitating careful consideration of the expression environment.