Recombinant Methanococcus maripaludis UPF0200 protein MMP1282 (MMP1282)

What is Methanococcus maripaludis and why is it significant for protein research?

Methanococcus maripaludis is a methanogenic archaeon originally isolated from salt marsh sediments. This hydrogenotrophic methanogen generates methane from hydrogen and carbon dioxide or formate. Its significance as a research model stems from its relatively rapid growth, available genetic tools, and complete genome sequence . M. maripaludis has emerged as a promising host organism for metabolic engineering of CO2-fixation pathways, making it valuable for studying archaeal proteins and their functions .

What expression systems are available for producing recombinant MMP1282 in M. maripaludis?

For heterologous protein expression in M. maripaludis, researchers can use the CRISPR/Cas12a toolbox that enables stable chromosomal integration of genes. This system allows for efficient knock-in of genes with a success rate of up to 95% . Several promoters have been characterized for protein expression in M. maripaludis, including strong constitutive promoters such as Pmcr, Pmcr_JJ, and Pfla_JJ from M. maripaludis JJ, as well as PglnA and Pmtr from Methanococcus vannielii SB . For MMP1282 expression, selecting the appropriate promoter depends on your experimental requirements - whether constitutive or regulated expression is desired.

How do I design primers for amplifying the MMP1282 gene from M. maripaludis genomic DNA?

When designing primers for MMP1282 amplification, consider the following methodological approach: (1) obtain the gene sequence from genome databases; (2) design primers with appropriate restriction sites compatible with your destination vector; (3) include 6-9 extra nucleotides upstream of restriction sites to ensure efficient enzyme cutting; (4) check primers for self-complementarity and secondary structure formation; and (5) optimize annealing temperatures based on GC content. Since M. maripaludis has a PstI restriction modification system, avoid including unmethylated PstI sites in your primers as these can reduce transformation efficiency by 1.6-3.4 fold per site .

What genetic tools are available for studying MMP1282 function in M. maripaludis?

Several genetic tools are available for studying protein function in M. maripaludis:

• CRISPR/Cas12a genome-editing toolbox: Enables efficient gene deletion or modification with success rates up to 95%, despite the hyperpolyploidy of M. maripaludis .

• Markerless mutagenesis system: Uses negative selection with the hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to 8-azahypoxanthine .

• Integration at the upt locus: Allows stable incorporation of constructs into the genome at the uracil phosphoribosyltransferase gene site .

• Homology-directed repair: Utilizes the endogenous homology-directed repair machinery in M. maripaludis for gene editing .

These tools provide versatile approaches for creating knockout strains, generating point mutations, or integrating reporter fusions to study MMP1282 function.

How can I create a knockout strain to study MMP1282 function?

To create an MMP1282 knockout strain using the CRISPR/Cas12a system, follow this methodology:

• Design a guide RNA (gRNA) targeting the MMP1282 gene using appropriate design tools to ensure specificity.

• Construct a repair fragment (RF) with homology arms of at least 500-1000 bp flanking the target region.

• Clone these components into the pMM002P plasmid, which contains the LbCas12a gene, the designed gRNA, and the repair fragment .

• Transform M. maripaludis with this construct, where the CRISPR/Cas12a system will create a double-stranded break in the MMP1282 gene.

• Select transformants and verify gene deletion through PCR amplification and sequencing.

This approach has demonstrated high efficiency with positive rates of 89-100% for gene editing in M. maripaludis .

What is the optimal homology arm length for targeted genetic modifications of MMP1282?

Based on experimental evidence with the CRISPR/Cas12a system in M. maripaludis, homology arms of 500-1000 bp provide optimal efficiency for genome editing. Studies have shown high positive rates (89-100%) with homology arms of these lengths . When designing homology arms for MMP1282 modification, consider:

• Using 500 bp homology arms as a standard starting point, as they provide a good balance between cloning ease and recombination efficiency.

• Extending to 1000 bp if initial attempts are unsuccessful or if modifying regions with lower recombination frequencies.

• Avoiding sequences containing PstI sites when possible, as these can reduce transformation efficiency due to M. maripaludis' restriction modification system .

• Ensuring the homology arms are within 1000 bp distance of the double-stranded break site for optimal repair efficiency .

What are the optimal conditions for expressing recombinant MMP1282 in M. maripaludis?

For optimal expression of recombinant MMP1282 in M. maripaludis, consider these methodological parameters:

• Promoter selection: Among the 15 different promoters characterized in M. maripaludis, strong constitutive promoters like PglnA, Pmtr, Pmcr, Pmcr_JJ, and Pfla_JJ provide high expression levels . For regulated expression, the Pnif promoter can be used in combination with nitrogen source manipulation or nrpR deletion to achieve very high expression levels (2670 ± 58 nmol min⁻¹ OD600⁻¹) .

• Growth substrate: Expression levels vary depending on whether formate or H2/CO2 is used as a growth substrate. Some promoters like PhdrC1 drive expression only in formate-containing growth medium .

• Integration site: Stable chromosomal integration at neutral loci, such as the upt gene site, ensures consistent expression without the need for continuous selection pressure .

• Growth temperature: Standard cultivation at 37°C provides a balance between growth rate and protein folding.

• Growth phase: Harvesting cells in mid-to-late exponential phase typically yields the highest protein concentrations.

What purification strategy is most effective for isolating recombinant MMP1282?

An effective purification strategy for archaeal proteins like MMP1282 involves:

• Cell lysis: Gentle lysis methods using non-ionic detergents or osmotic shock to preserve protein structure.

• Initial clarification: Centrifugation at 15,000-20,000 × g to remove cell debris, followed by ultracentrifugation if membrane-associated fractions need to be separated.

• Affinity chromatography: Using His-tag purification as a primary step, with optimized imidazole concentrations for binding and elution.

• Secondary purification: Ion exchange chromatography based on MMP1282's predicted isoelectric point.

• Polishing step: Size exclusion chromatography to remove aggregates and ensure homogeneity.

• Buffer optimization: Testing various buffers with different pH values and salt concentrations to maximize stability.

For archaeal proteins like MMP1282, including stabilizing agents such as glycerol (10-20%) and reducing agents may improve yield and activity during purification.

How can I verify the proper folding and activity of purified recombinant MMP1282?

To verify proper folding and activity of purified MMP1282, employ these methodological approaches:

• Structural integrity assessment:

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

  • Thermal shift assays to determine protein stability and folding state

  • Size exclusion chromatography to detect aggregation or oligomerization states

  • Limited proteolysis to assess compact folding

• Functional verification:

  • Since MMP1282 is a UPF0200 family protein with uncertain function, compare its properties with characterized homologs

  • Assess binding to potential cofactors using isothermal titration calorimetry or fluorescence-based assays

  • Conduct substrate screening using activity-based protein profiling

• Post-translational modification analysis:

  • Mass spectrometry to detect any archaeal-specific modifications

  • Phosphorylation status verification using ProQ Diamond staining or phospho-specific antibodies

• Protein-protein interaction studies:

  • Pull-down assays with native M. maripaludis extracts to identify interaction partners

  • Crosslinking mass spectrometry to map interaction interfaces

How can I conduct structure-function analysis of MMP1282?

For comprehensive structure-function analysis of MMP1282, implement these methodological approaches:

• Structural determination:

  • X-ray crystallography: Optimize crystallization conditions specifically for archaeal proteins, considering salt concentration and temperature effects

  • Cryo-EM: Particularly useful if MMP1282 forms larger complexes or is difficult to crystallize

  • NMR spectroscopy: For analyzing dynamic regions and ligand interactions

• Computational analysis:

  • Homology modeling based on structurally characterized UPF0200 family proteins

  • Molecular dynamics simulations to understand conformational flexibility

  • Docking studies to predict potential ligand binding sites

• Mutational analysis:

  • Use the CRISPR/Cas12a system to generate site-directed mutations in the native gene

  • Create an in-frame deletion series to identify functional domains

  • Complementation studies with mutated versions integrated at the upt site

• Functional validation:

  • Phenotypic analysis of mutant strains under various growth conditions

  • Biochemical assays based on predicted function

  • In vivo crosslinking to capture transient interactions

What considerations are important when designing experiments to characterize protein-protein interactions involving MMP1282?

When designing experiments to characterize protein-protein interactions involving MMP1282, consider these methodological considerations:

• Experimental approaches suitable for archaeal systems:

  • Co-immunoprecipitation using antibodies against MMP1282 or epitope tags

  • Proximity-labeling methods adapted for archaeal cells (BioID or APEX2)

  • Bacterial two-hybrid systems (archaeal proteins often express poorly in yeast)

  • Label-free quantitative proteomics comparing wild-type and MMP1282 knockout strains

• Validation strategies:

  • Reciprocal pull-downs with identified interaction partners

  • Co-localization studies using fluorescently tagged proteins

  • Genetic interaction studies using the CRISPR/Cas12a system to create double mutants

  • Biochemical reconstitution of protein complexes with purified components

• Archaeal-specific considerations:

  • Optimize buffers to maintain archaeal protein stability (higher salt concentrations, reducing conditions)

  • Consider potential post-translational modifications unique to archaea

  • Account for membrane association or compartmentalization in experimental design

• Controls:

  • Include unrelated archaeal proteins of similar size/charge as negative controls

  • Verify that tagging does not disrupt protein function through complementation studies

  • Use non-specific IgG controls for immunoprecipitation experiments

How do I optimize CRISPR/Cas12a-based genetic manipulation for studying MMP1282 regulatory networks?

To optimize CRISPR/Cas12a-based genetic manipulation for studying MMP1282 regulatory networks, implement this methodological framework:

• Guide RNA design optimization:

  • Design multiple gRNAs targeting different regions of MMP1282 and potential regulatory genes

  • Test gRNA efficiency using transformation efficiency as a proxy (lower transformation efficiency indicates effective DNA cleavage)

  • For regulatory studies, target both upstream and downstream regions to identify potential regulatory elements

• Repair template optimization:

  • Use homology arms of 500-1000 bp for highest efficiency

  • Position homology arms within 1000 bp of the double-stranded break site

  • For studying promoter elements, design repair templates that integrate reporter genes like β-glucuronidase (uidA)

• Multiplexed editing strategies:

  • Exploit LbCas12a's ability to process multiple gRNAs from a single transcript

  • Target multiple components of potential regulatory networks simultaneously

  • Design experiments to create deletion series or regulatory element swaps

• Phenotypic analysis pipeline:

  • Develop high-throughput screening methods relevant to MMP1282's predicted function

  • Compare transcriptomic profiles between wild-type and mutant strains

  • Implement metabolomic analysis to identify pathway perturbations

• Efficiency considerations:

  • The system has demonstrated positive rates of up to 95% for gene editing despite M. maripaludis hyperpolyploidy

  • Consider the PstI restriction modification system when designing constructs, as it can reduce transformation efficiency

How does MMP1282 compare to homologous proteins in other archaeal species?

To conduct comparative analysis of MMP1282 with homologs in other archaeal species, apply these methodological approaches:

• Phylogenetic analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Identify orthologs versus paralogs across archaeal lineages

  • Analyze selective pressure using dN/dS ratios to identify conserved functional residues

• Structural comparison:

  • Align MMP1282 with structurally characterized homologs

  • Identify conserved motifs and potential functional domains

  • Model structural differences that might indicate functional divergence

• Genomic context analysis:

  • Examine gene neighborhoods across different archaeal genomes

  • Identify conserved operonic structures that might indicate functional relationships

  • Compare regulatory elements across species

• Horizontal gene transfer assessment:

  • Similar to the alanine dehydrogenase, alanine racemase, and alanine permease genes in M. maripaludis that were acquired through lateral gene transfer from low-G+C gram-positive bacteria , investigate whether MMP1282 shows evidence of horizontal acquisition

• Experimental validation:

  • Express homologs from different species in M. maripaludis and test functional complementation

  • Use the CRISPR/Cas12a system to swap domains between homologs to identify functionally important regions

What metabolic pathways might involve MMP1282 based on genomic context and expression patterns?

To identify potential metabolic pathways involving MMP1282, implement these methodological approaches:

• Genomic context analysis:

  • Analyze genes adjacent to MMP1282 for functional clues

  • Identify potential operonic structures or co-regulated gene clusters

  • Compare with similar genomic arrangements in related methanogens

• Transcriptomic profiling:

  • Analyze expression patterns under different growth conditions (formate vs. H2/CO2 as growth substrates)

  • Compare expression with known metabolic genes to identify co-regulation patterns

  • Use nitrogen limitation or other stress conditions to identify regulatory relationships

• Metabolic reconstruction:

  • Map MMP1282 to the M. maripaludis metabolic network

  • Identify metabolic bottlenecks where UFP0200 family proteins might function

  • Consider potential roles in methanogenesis, nitrogen metabolism, or CO2 fixation

• Comparative analysis approach:

  • Similar to the analysis of alanine metabolism genes in M. maripaludis , investigate whether MMP1282 might be involved in unusual metabolic capabilities

  • Consider potential roles in substrate utilization, stress response, or energy conservation

• Experimental validation:

  • Create MMP1282 knockout using CRISPR/Cas12a and analyze growth on different substrates

  • Perform metabolomic analysis comparing wild-type and knockout strains

  • Conduct isotope labeling studies to track specific metabolic fluxes

What are common challenges in working with archaeal proteins like MMP1282 and how can they be overcome?

Common challenges in working with archaeal proteins like MMP1282 include:

• Protein expression challenges:

  • Challenge: Low expression yields in heterologous systems

  • Solution: Use homologous expression in M. maripaludis with optimized promoters like PglnA, Pmtr, Pmcr, Pmcr_JJ, or Pfla_JJ

  • Alternative: For regulated high expression, use the Pnif promoter in an nrpR deletion background (achieving up to 2670 ± 58 nmol min⁻¹ OD600⁻¹)

• Protein stability issues:

  • Challenge: Archaeal proteins often require specific buffer conditions

  • Solution: Optimize salt concentration, pH, and include stabilizing agents

  • Methodology: Perform thermal shift assays to identify optimal buffer conditions

• Genetic manipulation difficulties:

  • Challenge: M. maripaludis is polyploid, making complete gene deletion challenging

  • Solution: Use the CRISPR/Cas12a system, which achieves up to 95% efficiency despite polyploidy

  • Consideration: Position homology arms within 1000 bp of double-stranded breaks

• Functional characterization uncertainty:

  • Challenge: UPF0200 family proteins have poorly characterized functions

  • Solution: Combine computational predictions with broad-spectrum activity assays

  • Approach: Create reporter fusions to monitor expression under different conditions

• Anaerobic culture requirements:

  • Challenge: M. maripaludis requires strict anaerobic conditions

  • Solution: Develop efficient anaerobic workflows for protein purification

  • Methodology: Consider oxygen-tolerant purification approaches when possible

How can I resolve inconsistent results when comparing in vitro and in vivo studies of MMP1282?

To resolve inconsistencies between in vitro and in vivo studies of MMP1282, implement this methodological framework:

• Systematic validation approach:

  • Create multiple genetic controls, including clean deletions, point mutations, and complemented strains using the CRISPR/Cas12a system

  • Test protein function under varying buffer conditions to identify environmentally sensitive activities

  • Verify protein folding and oligomeric state both in vitro and in cell extracts

• Activity reconstitution strategy:

  • Identify potential missing cofactors or interaction partners from cell extracts

  • Test activity in the presence of cellular fractions or potential metabolites

  • Consider post-translational modifications present in vivo but absent in recombinant preparations

• Environmental parameter optimization:

  • Match in vitro conditions to intracellular environment (pH, salt concentration, reducing potential)

  • Test temperature-dependent activity profiles

  • Consider pressure effects if relevant to M. maripaludis' natural environment

• Analytical alignment:

  • Use identical detection methods for both in vitro and in vivo experiments

  • Develop internal controls and standard curves specific to each experimental system

  • Apply statistical methods to determine significance of observed differences

• Protein engineering approach:

  • Create tagged versions for in vivo localization and compare with biochemical fractionation

  • Use the native promoter and terminator for complementation studies to maintain natural expression levels

  • Consider allosteric regulation that may be disrupted in purified systems

How can studying MMP1282 contribute to our understanding of archaeal metabolism?

Studying MMP1282 can contribute to our understanding of archaeal metabolism through these research avenues:

• Methanogenesis pathway insights:

  • As M. maripaludis is a hydrogenotrophic methanogen that generates methane from hydrogen and carbon dioxide or formate , investigating MMP1282's potential role in this process could reveal novel regulatory mechanisms

  • Methodological approach: Compare metabolic fluxes in wild-type versus MMP1282 knockout strains using isotope labeling

• CO2 fixation applications:

  • M. maripaludis is recognized as a promising host organism for metabolic engineering of CO2-fixation pathways

  • Research direction: Explore whether MMP1282 influences carbon assimilation efficiency or regulation

• Archaeal-specific adaptations:

  • UPF0200 family proteins may represent archaeal adaptations to extreme environments

  • Experimental approach: Characterize MMP1282 stability and activity under various stress conditions

• Evolutionary insights:

  • Similar to the lateral gene transfer observed with alanine metabolism genes in M. maripaludis , MMP1282 study may reveal horizontal gene transfer events that shaped archaeal metabolism

  • Analytical method: Conduct comprehensive phylogenetic analysis across domains of life

• Novel metabolic capabilities:

  • M. maripaludis has unusual metabolic capabilities, such as utilizing both L- and D-alanine as nitrogen sources

  • Investigation approach: Test MMP1282 knockout strains for growth on various substrates to identify phenotypes

What potential biotechnological applications might arise from characterizing MMP1282?

Potential biotechnological applications from characterizing MMP1282 include:

• Biocatalyst development:

  • If MMP1282 possesses enzymatic activity, it may function under conditions that make it valuable for industrial processes

  • Application strategy: Engineer MMP1282 for enhanced stability or modified substrate specificity

• Methane production optimization:

  • Understanding MMP1282's role could lead to enhanced methane production in bioreactor systems

  • Engineering approach: Overexpress MMP1282 using strong promoters like PglnA or the derepressed Pnif system

• CO2 capture technology:

  • M. maripaludis is promising for CO2-fixation pathways , and MMP1282 characterization may contribute to this application

  • Development pathway: Integrate MMP1282 knowledge into metabolic models for strain optimization

• Archaeal expression system improvement:

  • Insights from MMP1282 expression could enhance M. maripaludis as a protein production platform

  • Methodological advancement: Apply the CRISPR/Cas12a toolbox for precise genetic manipulation

• Novel antimicrobial targets:

  • If MMP1282 represents a conserved archaeal protein with essential functions, homologs in pathogenic archaea could become therapeutic targets

  • Research direction: Conduct comparative analysis across clinically relevant archaeal species

• Extremozyme discovery:

  • UPF0200 family proteins may possess unique properties suitable for industrial applications

  • Characterization approach: Test stability and activity under extremes of temperature, pH, and salt concentration