Recombinant Neisseria gonorrhoeae Peptide chain release factor 1 (prfA)

What is Peptide chain release factor 1 (prfA) in Neisseria gonorrhoeae?

Peptide chain release factor 1 (prfA) in Neisseria gonorrhoeae is a critical protein involved in translation termination during protein synthesis. The protein functions by recognizing stop codons (UAA and UAG) in messenger RNA and catalyzing the hydrolysis of the peptidyl-tRNA bond, which releases the newly synthesized peptide chain. In N. gonorrhoeae strain NCCP11945, prfA is encoded by the gene identified with UniProt accession number B4RN52 . The protein consists of 358 amino acids forming a full-length protein with a specific sequence that includes conserved domains necessary for its function in bacterial translation.

What is the molecular structure of recombinant N. gonorrhoeae prfA?

Recombinant N. gonorrhoeae prfA is a full-length protein comprising 358 amino acids. According to available data, its amino acid sequence includes multiple functional domains responsible for stop codon recognition, peptidyl-tRNA interaction, and ribosome binding . While a crystal structure specifically for N. gonorrhoeae prfA has not been identified in the provided search results, research methodologies similar to those used for the Neisseria gonorrhoeae adhesin complex protein (Ng-ACP), which was solved at 1.65 Å resolution , could potentially be applied to determine the structural characteristics of prfA.

How stable is recombinant N. gonorrhoeae prfA under laboratory conditions?

The stability of recombinant N. gonorrhoeae prfA depends significantly on storage conditions. According to product specifications, the protein can be stored at -20°C for regular storage, but extended storage should be at -20°C or -80°C . The shelf life varies based on formulation: liquid formulations typically maintain stability for approximately 6 months at -20°C/-80°C, while lyophilized forms can remain stable for up to 12 months at the same temperatures . Repeated freeze-thaw cycles should be avoided to maintain protein integrity. Working aliquots can be stored at 4°C for up to one week .

What experimental approaches are recommended for reconstitution of recombinant N. gonorrhoeae prfA?

For optimal reconstitution of recombinant N. gonorrhoeae prfA, the following methodology is recommended:

• Begin by briefly centrifuging the vial to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Aliquot the reconstituted protein for long-term storage at -20°C/-80°C

This methodology helps maintain protein stability and prevents degradation during storage periods. The addition of glycerol serves as a cryoprotectant to prevent damage from freeze-thaw cycles.

How can researchers verify the purity and activity of recombinant N. gonorrhoeae prfA?

Researchers can verify the purity of recombinant N. gonorrhoeae prfA using SDS-PAGE analysis, with commercial preparations typically showing >85% purity . For activity assessment, researchers can employ in vitro translation termination assays that measure the protein's ability to recognize stop codons and catalyze peptidyl-tRNA hydrolysis. Similar to methodologies used for other N. gonorrhoeae proteins, Western blotting techniques can be used to confirm the identity and expression of the protein . Functional assays should be designed to test the protein's specific activity in releasing nascent peptide chains from ribosomes at stop codons.

What expression systems yield high-quality recombinant N. gonorrhoeae prfA?

Mammalian cell expression systems have been successfully used to produce recombinant N. gonorrhoeae prfA . This expression system likely provides proper folding and potential post-translational modifications that might be important for structural integrity. For other N. gonorrhoeae proteins like Ng-ACP, researchers have successfully used recombinant expression systems that yielded proteins capable of inducing functional antibodies, suggesting that properly folded, biologically active recombinant proteins can be produced . The choice of expression system should be guided by the intended experimental use, with mammalian systems potentially offering advantages for structural and functional studies.

How does N. gonorrhoeae prfA compare to peptide release factors in other pathogenic bacteria?

While the search results don't provide direct comparative data for prfA across different bacterial species, general principles of bacterial release factors suggest that N. gonorrhoeae prfA likely shares conserved functional domains with other bacterial RF1 proteins while potentially containing species-specific variations. Similar comparative approaches have been used with other N. gonorrhoeae proteins, such as the adhesin complex protein (ACP), which showed structural homology to Neisseria meningitidis ACP and MliC/PliC lysozyme inhibitors from other bacteria . Understanding these similarities and differences could provide insights into potential species-specific functions or therapeutic targeting strategies.

What is the potential role of prfA in N. gonorrhoeae pathogenesis and antibiotic resistance?

As a critical factor in protein synthesis termination, prfA likely plays an essential role in N. gonorrhoeae viability and potentially in pathogenesis through its impact on the expression of virulence factors. While the search results don't directly address prfA's role in antibiotic resistance, research on other N. gonorrhoeae proteins has shown connections between protein expression and pathogenicity. For example, lytic transglycosylases LtgA and LtgD have been shown to affect cytokine production and immune response during infection . Similar investigations into prfA could reveal its potential contributions to bacterial survival, virulence factor expression, and response to antibiotic treatments.

How might structural analysis of prfA contribute to therapeutic development against N. gonorrhoeae?

Structural analysis of N. gonorrhoeae prfA could significantly contribute to therapeutic development strategies. By determining the crystal structure through methodologies similar to those used for Ng-ACP (solved at 1.65 Å) , researchers could identify potential binding sites for small molecule inhibitors that could disrupt protein synthesis in N. gonorrhoeae. This approach is particularly relevant given that N. gonorrhoeae is listed by the World Health Organization as a high-priority pathogen for research and development of new control measures . The success of structural approaches with Ng-ACP, which led to identification of potential vaccine candidates , suggests that similar strategies could be productive for prfA.

What controls should be included when studying recombinant N. gonorrhoeae prfA activity?

When designing experiments to study recombinant N. gonorrhoeae prfA activity, researchers should include the following controls:

• Negative controls:

  • Heat-inactivated prfA to demonstrate specificity of activity

  • Buffer-only samples to establish baseline measurements

  • Non-cognate stop codon substrates to confirm codon specificity

• Positive controls:

  • Well-characterized peptide release factors from model organisms (e.g., E. coli RF1)

  • Synthetic peptidyl-tRNA substrates with known release kinetics

• Specificity controls:

  • RF2 protein (which recognizes UGA and UAA) to distinguish between RF1 and RF2 activities

  • Mutation controls with alterations in key functional residues

This multi-control approach enables rigorous validation of experimental results and helps distinguish prfA-specific effects from background or non-specific activities.

How can researchers investigate the interaction between prfA and the N. gonorrhoeae ribosome?

To investigate prfA-ribosome interactions in N. gonorrhoeae, researchers could employ the following methodological approaches:

• Co-immunoprecipitation assays to isolate prfA-ribosome complexes

• Cryo-electron microscopy to visualize structural interactions at near-atomic resolution

• Cross-linking mass spectrometry to identify specific contact points between prfA and ribosomal components

• Surface plasmon resonance or bio-layer interferometry to quantify binding kinetics

• Ribosome profiling to assess prfA activity in cells under different conditions

These approaches could provide complementary data about both structural and functional aspects of the prfA-ribosome interaction, similar to how structural and functional studies have been combined to understand other N. gonorrhoeae proteins like Ng-ACP .

What mutagenesis approaches would be most informative for studying N. gonorrhoeae prfA function?

Strategic mutagenesis approaches for studying N. gonorrhoeae prfA function would include:

• Alanine scanning mutagenesis of conserved domains to identify essential residues

• Targeted mutations in stop codon recognition domains to alter codon specificity

• Creation of chimeric proteins with RF1 from other species to identify species-specific functional regions

• Domain swapping with RF2 to understand class-specific functions

• Introduction of mutations corresponding to those that confer antibiotic resistance in other bacteria

The effectiveness of mutagenesis approaches has been demonstrated in studies of other N. gonorrhoeae proteins, such as the examination of LtgA and LtgD mutants and their effects on peptidoglycan release and immune response .

How does prfA function relate to N. gonorrhoeae immune evasion mechanisms?

While the search results don't directly address prfA's role in immune evasion, the protein synthesis machinery it belongs to likely contributes to the expression of virulence factors involved in immune evasion. Research on other N. gonorrhoeae proteins has revealed sophisticated immune modulation mechanisms. For example, the Ng-ACP protein has been shown to inhibit human lysozyme activity, a key component of innate immunity . Studies on LtgA and LtgD have demonstrated their impact on cytokine production and NOD1/NOD2 activation . Similar comprehensive studies of prfA could reveal connections between translation termination efficiency and the expression of immune evasion factors.

Could prfA be a potential target for novel antimicrobial strategies against N. gonorrhoeae?

As an essential factor in bacterial protein synthesis, prfA represents a potential target for novel antimicrobial strategies against N. gonorrhoeae. Given the increasing antibiotic resistance in N. gonorrhoeae and its designation by the WHO as a high-priority pathogen , new therapeutic approaches are urgently needed. Targeting prfA could potentially disrupt protein synthesis in a species-specific manner if structural or functional differences from human release factors can be identified and exploited. This approach aligns with broader efforts to develop new control measures against N. gonorrhoeae, as evidenced by research into vaccine candidates like Ng-ACP .

What are the primary challenges in purifying functionally active recombinant N. gonorrhoeae prfA?

The primary challenges in purifying functionally active recombinant N. gonorrhoeae prfA likely include:

• Maintaining proper folding during expression and purification

• Preserving activity during concentration and storage steps

• Ensuring removal of contaminating nucleases and proteases

• Achieving sufficient yield for structural and functional studies

• Confirming that the recombinant protein accurately represents native activity

To address these challenges, researchers can implement strategies such as:

• Optimization of expression conditions (temperature, induction parameters)

• Addition of stabilizing agents during purification

• Implementation of multiple chromatography steps to enhance purity

• Inclusion of activity assays at each purification stage

• Careful control of pH and salt conditions throughout the purification process

Similar challenges have been addressed in the production of other recombinant N. gonorrhoeae proteins for structural and functional studies .

How can researchers differentiate between effects of prfA inhibition and other translation disruptions?

To differentiate between specific prfA inhibition effects and broader translation disruption, researchers should employ a multi-faceted experimental approach:

This differentiated approach would allow researchers to attribute observed phenotypes specifically to prfA function rather than to general translation defects, providing more precise insights into the role of prfA in N. gonorrhoeae biology.