Recombinant Lactobacillus plantarum 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase (metE), partial

What is 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase (metE) and what is its fundamental role in L. plantarum metabolism?

5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase (metE) is an enzyme with EC number 2.1.1.14 that functions as a cobalamin-independent methionine synthase in L. plantarum. It catalyzes the transfer of a methyl group from 5-methyltetrahydropteroyltriglutamate to L-homocysteine, resulting in the formation of methionine. This reaction is crucial for the methionine biosynthesis pathway in bacteria that lack the cobalamin-dependent methionine synthase. In L. plantarum, particularly strain ATCC BAA-793/NCIMB 8826/WCFS1, this enzyme plays a vital role in amino acid metabolism and protein synthesis. The enzyme is also alternatively known as "Methionine synthase, vitamin-B12 independent isozyme," highlighting its function independent of vitamin B12 (cobalamin) .

What are the structural characteristics of recombinant L. plantarum metE?

Recombinant L. plantarum metE is typically produced as a partial protein with high purity (>85% as determined by SDS-PAGE). The recombinant protein maintains the catalytic domains necessary for its methyltransferase activity while potentially lacking some regions of the native enzyme. The protein's structure includes binding sites for its substrates (5-methyltetrahydropteroyltriglutamate and homocysteine) and likely contains conserved domains typical of methionine synthases. When expressed recombinantly, the protein may include additional tags determined during the manufacturing process, which can affect its structural properties but are designed to minimize interference with enzymatic function .

How do researchers differentiate between native and recombinant metE in experimental analyses?

Researchers can differentiate between native and recombinant metE through several analytical approaches. First, recombinant metE may contain fusion tags (determined during the manufacturing process) that are not present in the native enzyme, allowing for detection using tag-specific antibodies or assays. Second, recombinant metE expressed in E. coli, as is common with commercial preparations, may exhibit slight differences in post-translational modifications compared to native L. plantarum metE. Third, researchers can use strain-specific PCR primers targeting the recombinant construct to differentiate between native and introduced genes. For more precise differentiation, mass spectrometry can identify unique peptides resulting from the recombinant structure. Finally, comparative enzymatic activity assays may reveal differences in kinetic parameters between native and recombinant forms .

What expression systems are most effective for producing recombinant L. plantarum metE?

For the recombinant production of L. plantarum metE, several effective expression systems have been documented. The pSIP expression system has demonstrated particular efficacy for producing recombinant proteins in L. plantarum. This system, which has been successfully used for various proteins from L. plantarum WCFS1, allows for inducible expression and has been optimized for both intracellular expression and protein secretion. While the search results do not specifically mention metE expression with this system, the pSIP401 and pSIP409 vectors have shown success with other L. plantarum enzymes .

For recombinant metE production, E. coli-based expression systems are commonly employed due to their high yield and established protocols, as evidenced by commercial recombinant metE being sourced from E. coli. When choosing an expression system, researchers should consider factors such as required post-translational modifications, protein solubility, and the intended application of the recombinant protein. For studies requiring native-like activity, expressing metE in a L. plantarum host using homologous recombination or suitable expression vectors may preserve functional characteristics better than heterologous expression systems .

What are the optimal conditions for storage and handling of recombinant L. plantarum metE to maintain activity?

The optimal storage conditions for recombinant L. plantarum metE vary based on the preparation format. For liquid formulations, stability is maintained for approximately 6 months when stored at -20°C to -80°C. Lyophilized (freeze-dried) preparations offer extended stability of up to 12 months when stored at similar temperatures (-20°C to -80°C). When working with the protein, repeated freeze-thaw cycles should be strictly avoided as they significantly compromise enzymatic activity and structural integrity .

For short-term storage during active research, working aliquots can be maintained at 4°C for up to one week. When reconstituting lyophilized protein, it is recommended to first centrifuge the vial briefly to ensure all contents are at the bottom. The protein should be reconstituted in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. For long-term storage of reconstituted protein, adding glycerol to a final concentration of 5-50% (with 50% being typical) before aliquoting and storing at -20°C to -80°C significantly enhances stability .

What signal peptides optimize the secretion efficiency of recombinant proteins in L. plantarum expression systems?

The selection of appropriate signal peptides significantly impacts the secretion efficiency of recombinant proteins in L. plantarum. Research has identified several signal peptides derived from L. plantarum WCFS1 that demonstrate superior performance. Among these, Lp_2145, Lp_3050, and Lp_0373 have been extensively tested with recombinant proteins. For instance, when expressing the α-amylase AmyL from L. plantarum S21, the Lp_2145 signal peptide resulted in the highest total and extracellular enzymatic activities, with values of 13.1 and 8.1 kU/L of fermentation, respectively. This represented a substantial increase compared to expression using the native signal peptide (SP_AmyL), with 6.2-fold and 5.4-fold increases in total and extracellular activities .

For optimal secretion efficiency (defined as the ratio of extracellular to total protein), the Lp_0373 signal peptide demonstrated superior performance among non-cognate signal peptides. The efficiency of these signal peptides appears to be protein-specific, suggesting that screening multiple signal peptides may be necessary when working with novel recombinant proteins such as metE. It's important to note that L. plantarum utilizes the Sec secretion machinery (with the Twin-arginine translocation pathway being absent), requiring proteins to be translocated as unfolded polypeptides .

How can recombinant L. plantarum metE contribute to immunomodulation research?

Recombinant L. plantarum strains have demonstrated significant immunomodulatory properties that could be extended to metE-expressing strains in sophisticated research applications. L. plantarum has been shown to inhibit the development of allergic disorders through modulation of T-cell responses, specifically by suppressing both Th1 and Th2 cytokine production. In experimental models, recombinant L. plantarum expressing specific epitopes inhibited IFNγ production (a non-specific effect) and IL5 production (an antigen-specific effect), suggesting complex immunomodulatory capabilities .

Researchers investigating metE-expressing L. plantarum could explore potential immunomodulatory effects through several experimental approaches: First, measuring cytokine production profiles (both pro-inflammatory cytokines like TNF-α and IL-6 and anti-inflammatory cytokines like IL-10) in response to stimulation with the recombinant bacteria. Second, investigating T-cell polarization effects in vitro and in vivo using models similar to those employed with dust mite epitope-expressing recombinants. Third, evaluating potential adjuvant properties when co-administered with antigens of interest. These approaches would help elucidate whether metE-expressing L. plantarum offers unique immunomodulatory properties that could be exploited for therapeutic purposes .

What methodological approaches are recommended for studying the role of metE in one-carbon metabolism pathways of L. plantarum?

Investigating metE's role in one-carbon metabolism requires multiple complementary methodological approaches. First, researchers should consider gene knockout or CRISPR-based editing of the metE gene in L. plantarum, followed by metabolomic analysis to identify pathway alterations. This would reveal the direct and indirect metabolic consequences of metE absence. Stable isotope labeling experiments using 13C-labeled folate precursors or homocysteine would allow tracking of metabolic flux through the methionine synthesis pathway in wild-type versus metE-modified strains .

For functional characterization, in vitro enzymatic assays with purified recombinant metE should measure kinetic parameters (Km, Vmax) under various conditions to understand substrate specificity and regulatory mechanisms. RNA-Seq and RT-qPCR analyses can identify transcriptional changes in response to metE modulation, revealing potential regulatory networks. Additionally, researchers should perform comparative growth studies in media with and without methionine supplementation to establish the essentiality of metE under different nutritional conditions. These methodologies provide a comprehensive understanding of metE's metabolic significance in L. plantarum's one-carbon metabolism pathways .

What are the potential applications of recombinant L. plantarum metE in developing novel biotherapeutics?

Recombinant L. plantarum expressing metE offers several promising avenues for biotherapeutic development. The "Generally Regarded as Safe" (GRAS) status of L. plantarum makes it an attractive vector for delivering therapeutic proteins to mucosal surfaces. Researchers could exploit this by designing recombinant L. plantarum that co-expresses metE with therapeutic epitopes or antigens. The potential immunomodulatory properties of L. plantarum, combined with its ability to survive gastrointestinal conditions, make it particularly suitable for oral or mucosal administration of biotherapeutics .

One particularly promising application involves creating recombinant L. plantarum strains that express both metE and disease-specific epitopes for targeted immunomodulation. This approach has been successfully demonstrated with recombinant L. plantarum expressing SARS-CoV-2 epitopes, which induced significant immune responses when administered to animal models. The metE enzyme could potentially serve as a metabolic engineering tool to enhance survival and colonization of these therapeutic strains in vivo. Additionally, metE's role in methionine biosynthesis could be exploited to develop auxotrophic strains with enhanced safety profiles for clinical applications .

What are common challenges when working with recombinant L. plantarum metE and how can they be addressed?

Researchers working with recombinant L. plantarum metE frequently encounter several challenges. Protein solubility issues may arise during expression, particularly with the cobalamin-independent methionine synthase, which is a relatively large enzyme. This can be addressed by optimizing expression conditions (temperature, induction parameters) or using solubility-enhancing fusion tags. Another common challenge is variability in expression levels between experiments. This can be mitigated through rigorous standardization of culture conditions and by using RT-qPCR to monitor transcript levels, as demonstrated in studies with other L. plantarum recombinant proteins .

Stability concerns during purification and storage represent another significant challenge. To address this, researchers should follow strict storage recommendations, including maintaining appropriate temperature conditions (-20°C to -80°C) and adding glycerol (5-50%) to prevent activity loss. For purification challenges, optimizing buffer conditions and minimizing processing time can help maintain enzyme activity. Additionally, codon optimization may be necessary when expressing L. plantarum metE in heterologous hosts like E. coli to enhance expression efficiency and reduce the formation of truncated products .

How can researchers accurately quantify expression levels of recombinant metE in L. plantarum?

Accurate quantification of recombinant metE expression in L. plantarum requires multiple complementary approaches. Real-time reverse-transcriptase quantitative PCR (RT-qPCR) represents a highly sensitive method for measuring mRNA levels of the target gene. This technique has been successfully applied to other recombinant proteins in L. plantarum, where it revealed that different signal peptides can significantly affect transcript levels. When implementing RT-qPCR, researchers should carefully select appropriate reference genes for normalization and design primers specific to the recombinant metE construct .

At the protein level, quantification can be performed using enzyme activity assays specific to metE's methyltransferase function, providing functional data that correlates with expression levels. Western blot analysis with antibodies targeting either metE itself or fusion tags provides another quantification method and can distinguish between intracellular and secreted fractions. For more precise quantification, mass spectrometry-based approaches such as selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) can determine absolute protein concentrations. These methods, when used in combination, provide comprehensive data on both transcript and protein levels, enabling detailed analysis of expression efficiency .

What analytical methods are recommended for verifying the purity and activity of recombinant L. plantarum metE?

Verifying the purity and activity of recombinant L. plantarum metE requires a multi-faceted analytical approach. For purity assessment, SDS-PAGE remains the standard method, with commercial preparations typically achieving >85% purity. This can be complemented with more sensitive techniques like capillary electrophoresis or size-exclusion chromatography to detect minor contaminants. Western blotting with metE-specific antibodies can confirm the identity of the purified protein and detect potential degradation products .

For activity verification, enzyme-specific assays measuring the conversion of homocysteine to methionine provide direct evidence of functional integrity. These assays typically monitor either the consumption of substrates or the formation of methionine using spectrophotometric, HPLC, or coupled enzyme approaches. Thermal shift assays can assess protein stability under various conditions, helping optimize buffer compositions for maximum activity retention. Circular dichroism spectroscopy offers insights into secondary structure integrity, which correlates with enzymatic function. When implementing these methods, researchers should include appropriate positive controls and reference standards to ensure reliable interpretation of results .

How might emerging gene editing technologies enhance the study and application of recombinant L. plantarum metE?

Emerging gene editing technologies, particularly CRISPR-Cas systems, offer transformative opportunities for studying and manipulating L. plantarum metE. CRISPR-based approaches enable precise genomic modifications including targeted mutations, domain swapping, or regulatory element alterations that were previously challenging to achieve in L. plantarum. Researchers could create metE variants with modified substrate specificity or improved catalytic efficiency through directed evolution approaches combined with high-throughput screening. Additionally, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) systems could modulate metE expression without permanent genetic changes, allowing for temporal control of enzyme levels .

These technologies also facilitate the creation of optimized expression cassettes that integrate into specific genomic loci, potentially overcoming the variability often observed with plasmid-based expression systems. For vaccine development applications, precise genomic integration of metE alongside antigenic determinants could create stable recombinant strains with enhanced immunomodulatory properties. Furthermore, multiplex editing could simultaneously modify metE and related metabolic pathways to create strains with customized metabolic profiles optimized for specific biotechnological applications .

What are the potential synergistic effects of co-expressing metE with other recombinant proteins in L. plantarum?

Co-expression of metE with other recombinant proteins in L. plantarum could yield significant synergistic effects across multiple application domains. In metabolic engineering, co-expressing metE with other enzymes involved in methionine metabolism could create strains with enhanced production of sulfur-containing compounds valuable for food and pharmaceutical applications. The methionine biosynthetic pathway could be linked to other metabolic pathways to create novel biosynthetic routes for valuable compounds. From an immunomodulatory perspective, co-expression of metE with antigenic determinants or immunomodulatory cytokines could enhance vaccine efficacy by providing metabolic advantages to the bacterial vector while simultaneously delivering therapeutic proteins .

For protein production applications, co-expressing metE with chaperones or foldases could enhance the folding efficiency and stability of other recombinant proteins produced in the same system. This approach could be particularly valuable for difficult-to-express proteins. Additionally, co-expression with secretion-enhancing factors could improve protein export, building on the documented success of signal peptide optimization in L. plantarum. Experimental design for such co-expression studies should include careful analysis of metabolic burden, potential competition for cellular resources, and optimization of expression ratios to maximize synergistic benefits while minimizing antagonistic effects .