Recombinant Lactobacillus johnsonii Thymidylate kinase (tmk)

Introduction to Recombinant Lactobacillus johnsonii Thymidylate Kinase (Tmk)

Thymidylate kinase (Tmk) is an enzyme (EC= 2.7.4.9) also known as dTMP kinase, which plays a crucial role in DNA synthesis by catalyzing the phosphorylation of deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) . In Lactobacillus johnsonii, Tmk is essential for providing the necessary precursors for DNA replication and cell growth . Recombinant Lactobacillus johnsonii Tmk refers to the Tmk enzyme that is produced using recombinant DNA technology, where the gene encoding Tmk from L. johnsonii is expressed in a host organism, such as E. coli .

Production and Purification of Recombinant Tmk

The production of recombinant Tmk involves several key steps:

1. Gene Cloning and Expression Vector Construction: The tmk gene from Lactobacillus johnsonii is isolated and cloned into an expression vector. This vector is designed for high-level protein expression in a host organism like E. coli .

2. Transformation and Culture: The expression vector containing the tmk gene is transformed into E. coli cells. These cells are then cultured under conditions that promote the expression of the recombinant Tmk protein .

3. Protein Purification: After культивирования, the recombinant Tmk protein is purified from the E. coli lysate using various chromatography techniques, such as affinity chromatography, ion exchange chromatography, or size exclusion chromatography . The purity of the obtained protein is generally greater than 85% as determined by SDS-PAGE .

Table 1: Purification Steps and Expected Yield

Biochemical Characteristics of Recombinant Tmk

Recombinant Lactobacillus johnsonii Tmk exhibits typical enzymatic activity, catalyzing the phosphorylation of dTMP to dTDP using ATP as a phosphate donor. The enzyme's activity can be characterized by measuring the rate of dTMP phosphorylation under various conditions, including different pH levels, temperatures, and substrate concentrations.

Table 2: Biochemical Properties of Recombinant Tmk

Applications of Recombinant Lactobacillus johnsonii Tmk

1. Drug Discovery: Tmk is an attractive target for developing new antibacterial agents. Inhibitors of Tmk can disrupt DNA synthesis in bacteria, leading to cell death . Recombinant Tmk is used in high-throughput screening assays to identify and characterize potential Tmk inhibitors.

2. Structural Studies: Recombinant Tmk is used to determine the three-dimensional structure of the enzyme through X-ray crystallography or NMR spectroscopy. This structural information aids in the rational design of Tmk inhibitors .

3. Enzyme Mechanism Studies: Recombinant Tmk is used to study the enzyme's mechanism of action. By mutating specific amino acid residues in the active site and analyzing the effects on enzyme activity, researchers can gain insights into the catalytic mechanism of Tmk.

Genetic Engineering and Enhanced Functionality

Recombinant Lactobacillus johnsonii can be genetically engineered to express various proteins that offer protective effects against diseases . For example, L. johnsonii can be engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF), which reduces inflammation and promotes tissue repair .

• Anti-inflammatory Effects: Engineered L. johnsonii strains expressing GM-CSF have shown promise in reducing inflammation levels induced by E. coli infections. This is evidenced by decreased expression of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α .

• Improved Uterine Health: Studies have demonstrated that recombinant L. johnsonii strains can improve uterine morphology and reduce pathological damage in животные with endometritis, highlighting their potential in preventing postpartum uterine inflammation in bovines .

Table 3: Impact of Recombinant L. johnsonii on Inflammatory Markers

Role in Leucine Degradation

Recombinant bacteria, including modified Lactobacillus strains, can be engineered to degrade specific amino acids like leucine. This is achieved by introducing genetic circuits that express enzymes involved in leucine degradation pathways .

• Leucine Degradation Pathways: Enzymes such as keto-acid decarboxylase (KivD) and leucine dehydrogenase (LeuDH) play key roles in breaking down leucine into other metabolites .

• Enhanced Degradation: Overexpression of these enzymes, along with transporters like BrnQ, can significantly improve the rate of leucine degradation in bacterial strains .

Table 4: Enzymes and Transporters Involved in Leucine Degradation

Host-Directed Therapies

Recombinant Lactobacillus strains can also be used in host-directed therapies (HDT) to combat intracellular pathogens. These therapies focus on modulating host pathways to enhance the immune response against infections .

• Receptor Tyrosine Kinases (RTKs): Targeting RTKs with specific inhibitors has been shown to inhibit intracellular pathogens like Mycobacterium tuberculosis .

• Immunomodulation: Probiotic species like Lactobacillus reuteri produce compounds such as histamine that can modulate host mucosal immunity and suppress TNF production .