A system for the production of soluble Trypanosoma brucei gambiense phospholipase A2 (TbgPLA2)

Abstract

A system for the production of soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2), consisting of: a bioinformatics analysis unit configured to predict the 3D structure, topology, and signal peptide positions of TbgPLA2; a gene construct manufacturing unit configured to produce codon-optimized TbgPLA2 gene constructs with removed signal peptide and transmembrane sequences; an expression unit configured to express the TbgPLA2 constructs in host cells selected from the group consisting of E. coli and Pichia Pfarrer; a purification unit configured to purify TbgPLA2 from soluble fractions and inclusion bodies using nickel column metal affinity chromatography; and a refolding unit configured to renature TbgPLA2 using optimized buffer conditions consisting of Tris 55 mM, NaCl 21 mM, KCl 0.88 mM at pH 8.2.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2). More specifically, the present invention provides a system configured to produce soluble Trypanosoma Brucei gambiense phospholipase A2 through integrated units configured for bioinformatics analysis, preparation of gene constructs, expression in multiple host systems, purification, and protein refolding. BACKGROUND OF THE INVENTION

[0002] African trypanosomiasis is a major neglected tropical disease that affects both humans and animals in sub-Saharan Africa, putting approximately 70 million people at risk of infection. Human African trypanosomiasis (HAT), commonly known as sleeping sickness, is transmitted by tsetse flies during a blood meal of infected hosts. The economic consequences are significant: the estimated annual damage from trypanosomiasis in Africa is US$5 billion.

[0003] The disease continues to pose a major public health challenge due to the lack of effective vaccines and limited therapeutic options. Existing medications exhibit severe side effects, toxicity, and reduced efficacy due to the emergence of drug-resistant parasite strains. Trypanosomes exhibit remarkable adaptive capabilities, undergoing morphological changes and metabolic adaptations to survive in diverse host environments during their complex life cycle.

[0004] Phospholipase A2 (PLA2) plays a crucial role in the survival of trypanosomes by hydrolyzing membrane phospholipids at the sn-2 position, releasing arachidonic acid and lysophospholipids, which serve as precursors to important signaling molecules. TbgPLA2 is highly conserved across different trypanosome species but differs significantly from human homologs, making it an attractive drug target. The enzyme's essential role in parasite survival has been confirmed by studies showing that PLA 2- Inhibitors against both Trypanosoma Brucei brucei and Trypanosoma Brucei gambiense.

[0005] Despite its validation as a therapeutic target, the development of TbgPLA2-based interventions has been hampered by the ongoing challenge of producing soluble and enzymatically active forms of the enzyme. This creates a critical bottleneck in drug discovery and structural biology research. Therefore, there is an urgent need to develop soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2). To address this challenge, the present invention provides a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2). Summary of the invention

[0006] The present disclosure relates to a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2). The present invention relates to a comprehensive system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2) enzyme. The system integrates bioinformatics analysis, gene construct preparation, expression in multiple host systems, purification techniques, and protein refolding to address the long-standing challenge of producing active, soluble TbgPLA2. This represents the first successful, industrially scalable production system for soluble phospholipase A2 from a Trypanosoma species.

[0007] The present disclosure further relates to a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2). The system comprises: a bioinformatics analysis unit configured to predict the 3D structure, topology, and signal peptide positions of TbgPLA2; a gene construct production unit configured to produce codon-optimized TbgPLA2 gene constructs with removed signal peptide and transmembrane sequences; an expression unit configured to express the TbgPLA2 constructs in host cells selected from the group consisting of E.coli and Pichia pastoris; a purification unit configured to purify TbgPLA2 from soluble fractions and inclusion bodies using nickel column metal affinity chromatography; and a refolding unit configured to renature TbgPLA2 using optimized buffer conditions consisting of 55 mM Tris, 21 mM NaCl, and 0.88 mM KCl at pH 8.2.

[0008] An object of the present disclosure is to provide a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2).

[0009] Another aim of the present disclosure is the production of soluble and enzymatically active TbgPLA2, which can be used for drug research and therapy development against African trypanosomiasis.

[0010] Another aim of the present disclosure is to produce highly purified TbgPLA2 suitable for structural biology studies and 3D structure determination.

[0011] A further aim of the present disclosure is to enable the production of TbgPLA2 with high specific activity on an industrial scale for commercial applications, including the development of pharmaceutical products, the production of biodiesel and the manufacture of detergents.

[0012] To further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments illustrated in the accompanying drawings. These drawings merely illustrate typical embodiments of the invention and are therefore not to be considered as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings. SHORT DESCRIPTION OF THE FIGURE

[0013] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. 1 shows a block diagram of a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2) according to an embodiment of the present disclosure.

[0014] Those skilled in the art will also appreciate that the elements in the drawings are shown for convenience and are not necessarily to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, with respect to device construction, one or more components of the device may be represented in the drawings by conventional symbols. The drawing may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawing with details that would be readily apparent to those skilled in the art from the present description. DETAILED DESCRIPTION:

[0015] To facilitate understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and a clear description will be given. However, the scope of the invention is not limited thereby. Changes and further modifications to the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0017] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the language "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not refer to the same embodiment.

[0018] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps may include not only those steps, but also additional steps not expressly listed or inherent in that process or method. Likewise, the statement "comprises" for one or more devices, subsystems, elements, structures, or components does not exclude, without further limitation, the existence of other devices, subsystems, elements, structures, components, or additional devices, subsystems, elements, structures, or components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The systems, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. 1 shows a block diagram of a system (100) for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2), according to an embodiment of the present disclosure.

[0022] Referring to Fig.1, the system (100) comprises: a bioinformatics analysis unit (102) configured to predict the 3D structure, topology, and signal peptide positions of TbgPLA2; a gene construct production unit (104) configured to produce codon-optimized TbgPLA2 gene constructs with removed signal peptide and transmembrane sequences; an expression unit (106) configured to express the TbgPLA2 constructs in host cells selected from the group consisting of E. coli and Pichia pastoris; a purification unit (108) configured to purify TbgPLA2 from soluble fractions and inclusion bodies using nickel column metal affinity chromatography; and a refolding unit (110) configured to renature TbgPLA2 using optimized buffer conditions comprising Tris 55 mM, NaCl 21 mM, KCl 0.88 mM at pH 8.2.

[0023] In one embodiment, the gene construct production unit (104) is configured to produce constructs with N-terminal FLAG tag and C-terminal tags selected from the group consisting of 6xHis tag, EGFP tag and mClover tag.

[0024] In one embodiment, the expression unit (106) is configured to express TbgPLA2 in E. coli cells selected from the group consisting of RIL, RIPL, Arctic Express, pLysS and Rosetta 2 cells.

[0025] In one embodiment, the purification unit (108) is configured to solubilize inclusion bodies using 4 M urea and to purify both full-length 58 kDa TbgPLA2 and truncated mature 39 kDa TbgPLA2.

[0026] In one embodiment, the expression unit (106) is configured to express TbgPLA2 in Pichia pastoris using the pPICZ vector with Zeocin resistance marker.

[0027] In one embodiment, the system (100) further comprises a cleavage unit (112) configured to remove fusion tags via TEV cleavage and isolate free TbgPLA2 with a buffer containing 0.1% FOS-choline 12.

[0028] In one embodiment, the system (100) further comprises a characterization unit (114) configured to determine enzymatic activity parameters, wherein the TbgPLA2 has a Vmax of 25,126 µmol / min and a Km of 1,194 mg / ml.

[0029] In one embodiment, the gene construct production unit (104) is configured to produce a codon-optimized TbgPLA 2- Gene with a codon adaptation index of 0.9 and a GC content of 57%.

[0030] In one embodiment, the system (100) further comprises a validation unit (116) configured to verify TbgPLA2 expression by SDS-PAGE analysis, Western blotting with anti-His monoclonal antibody, and UV transillumination.

[0031] In one embodiment, the system (100) is configured to produce soluble TbgPLA2 with a specific activity of 107.14 µmol / min / mg for industrial applications selected from the group consisting of pharmaceutical applications, degumming agents, emulsifiers, detergent production, and biodiesel production.

[0032] The present invention relates to a system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2). The system operates with five integrated functional units that sequentially produce soluble TbgPLA2. The bioinformatics analysis unit first predicts the 3D structure and topology and identifies problematic signal peptides and transmembrane regions that lead to insolubility. This computational analysis guides the gene construct production unit, which creates optimized gene constructs from removed signal peptides and transmembrane sequences, while simultaneously incorporating affinity tags such as N-terminal FLAG tags and C-terminal His, EGFP, or mClover tags.

[0033] The expression unit utilizes dual host systems, namely E. coli (including specialized strains such as pLysS, Rosetta 2, RIL, RIPL, and Arctic Express) and Pichia pastoris, to meet diverse expression requirements. For protein expression in inclusion bodies, the purification unit uses metal affinity chromatography with nickel columns and 4M urea solubilization for inclusion body processing. The system can isolate both full-length TbgPLA2 (58 kDa) and truncated mature forms (39 kDa).

[0034] The refolding unit represents a key innovation and utilizes optimized buffer conditions (Tris 55 mM, NaCl 21 mM, KCl 0.88 mM at pH 8.2) identified through systematic screening. Additional units include a cleavage system for removing fusion markers using TEV protease and FOS-choline-12 buffer, as well as characterization features that confirm that the final product exhibits optimal enzymatic parameters (Vmax of 25.126 µmol / min, Km of 1.194 mg / ml) with a specific activity of 107.14 µmol / min / mg.

[0035] In one embodiment, the system for producing soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2) has a comprehensive design that combines structural bioinformatics analysis capabilities with gene construct generation capabilities. The bioinformatics analysis unit analyzes the TbgPLA 2 -Gene structure and generates optimized constructs for the production of soluble TbgPLA2. The system purifies the enzyme to homogeneity and enables the screening of nanobodies and compound libraries to discover therapeutic compounds that bind to or inhibit PLA2 activity. These screening capabilities form the basis for the development of potential therapeutics against TbgPLA2.

[0036] In one embodiment, the system has a comparative analysis function for evaluating PLA2 - Sequences of different trypanosome species compared to human PLA2 -Homologs. This unit of analysis demonstrates that PLA2 is highly conserved among trypanosome species, yet differs significantly from human homologs. The system exploits these differences to minimize toxicities and adverse effects, thus addressing the primary cause of drug failure and toxicity resulting from interactions with similar host molecules.

[0037] In one embodiment, the gene construct production unit is configured to obtain complete coding sequences (CDS) for TbgPLA2 from GenBank and perform codon optimization for optimal expression in Escherichia coli. The optimization system achieves a codon adaptation index of 0.9 and maintains the GC content of the codon-optimized gene at 57%. The gene synthesis and subcloning unit utilizes pET-24b expression vectors with kanamycin-resistant markers and C-terminal 6xHis tags and uses XhoI and NdeI restriction sites for cloning. The verification system confirms successful synthesis by Sanger sequencing and validates subcloning by agarose gel electrophoresis, with the codon-optimized TbgPLA2 gene migrating with a molecular weight of 1344 bp.

[0038] In one embodiment, the expression unit is configured to transform pET-24b-TbgPLA2 plasmids into chemically competent E. coli cells using heat shock transformation for heterologous expression. System evaluation revealed that expression attempts in RIL, RIPL, and Arctic Express E. coli cells failed due to protein toxicity. The purification unit includes functions for cell pellet harvesting, cell lysis, and purification using Ni-NTA columns. The system confirmed the absence of PLA2 in soluble fractions by Western blot analysis and subsequently configured inclusion body processing by solubilizing the pellets in 4 M urea, followed by nickel column metal affinity chromatography.The characterization unit identifies protein fragments by Coomassie staining, with confirmation by Ponceau staining and Western blotting with mouse monoclonal anti-His antibodies, establishing that expression cells cannot produce TbgPLA2 due to protein toxicity. The specialized expression unit contains pLysS expression cells with T7 lysosome systems for tight expression regulation and is therefore suitable for the expression of toxic proteins. With this configuration, the system successfully expresses TbgPLA2. The solubility test unit determines protein localization in inclusion bodies using RIPA buffer analysis. The purification system processes inclusion bodies by solubilizing pLysS expression pellets with 4 M urea. SDS-PAGE analysis reveals two prominent protein bands at 58 kDa (full-length) and 39 kDa (truncated mature protein).

[0039] In one embodiment, the refolding unit includes a comprehensive screening system with nine different buffer conditions to determine optimal renaturation parameters for TbgPLA2. The refolding system uses a drop dilution method with progress monitoring by SDS-PAGE analysis at 12-hour intervals to determine the timeframe for complete renaturation. The optimization system determines the optimal refolding buffer composition as Tris 55 mM, NaCl 21 mM, KCl 0.88 mM at pH 8.2. The validation unit confirms successful refolding through mobility shift assays using SDS-PAGE, where migration differences between reducing and non-reducing samples indicate complete protein renaturation. The immunoblot analysis system detects refolded proteins at the expected molecular weights using monoclonal mouse anti-6xHis antibodies.

[0040] In one embodiment, the alternative expression system uses Pichia pastoris with cloning of the TbgPLA2 gene into pPICZ vectors with Zeocin resistance markers, N-terminal FLAG tags, TEV sites, and C-terminal EGFP sequences in front of histidine tags. The system successfully expresses active whole protein, with localization in membrane fractions due to intact signal peptides and transmembrane helices. The purification unit processes whole TbgPLA2 protein from membrane fractions and verifies expression by SDS-PAGE and UV transillumination analysis.

[0041] In one embodiment, the system addresses the challenge of truncated 39 kDa insoluble protein production in pLysS expression, where signal peptides and transmembrane sequences are lost due to bacterial protease cleavage in vivo. The strategy involves removing signal peptides and transmembrane sequences and replacing them with N-terminal FLAG sequences. The structure modeling unit generates 3D structure predictions and topology analyses and identifies amino acid sequences in transmembrane helices using TMHMM and HMMTOP server analysis. The system identifies hydrophobic amino acid patches that interact with lipid bilayers at positions 137–154 and 177–196.

[0042] In one embodiment, the construct generation unit creates new constructs by removing signal peptides and transmembrane helices while retaining the C-terminal EGFP in pPICZ constructs for Pichia pastoris expression. System analysis shows weak expression in Pichia pastoris by SDS-PAGE band intensity evaluation. Additional construct preparation utilizes pFLAG vectors (derived from pMBP) for E. coli expression and generates constructs both with and without C-terminal mClover tags. The verification system confirms the successful cloning and induction of mClover fusion constructs by flow cytometry.

[0043] In one embodiment, the fluorescence monitoring system integrates EGFP and mClover additions into constructs for expression monitoring under UV irradiation and microscopy. The FLAG sequence serves as a scaffold mimicking transmembrane sequences and as an affinity marker for anti-FLAG antibody purification and Western blot detection. The expression and purification system utilizes Rosetta-2 cells with Ni-NTA purification capacity. The cleavage system removes mClover markers through overnight TEV cleavage to release free PLA2. After cleavage, TEV enzymes and cleaved fusion proteins are removed by nickel column loading, and free TbgPLA2 is collected in the column flowthrough.

[0044] In one embodiment, the system identifies TbgPLA2 aggregation tendencies with mClover and EGFP, which complicates complete removal of the fusion protein during nickel purification after TEV. The buffer optimization system contains 0.1% FOS-Choline 12 for partial removal of the fusion protein during small-scale purification, although large-scale applications remain challenging.

[0045] In one embodiment, the final optimization system addresses aggregation issues by removing mClover tags from E. coli expression constructs while retaining N-terminal FLAG tags. The refined expression system uses Rosetta 2 cells to produce soluble and active TbgPLA2, with purification and SDS-PAGE analysis confirming successful expression of 39 kDa mature TbgPLA2. The characterization system determines that TbgPLA2 released from fusion constructs has a specific activity of 54.54 µmol / min / mg, compared to 107.14 µmol / min / mg for constructs expressed without mClover tags. The enzymatic characterization unit found that the optimized system produced TbgPLA2 with Vmax of 25,126 µmol / min and Km of 1,194 mg / ml, demonstrating the successful production of enzymatically active soluble protein.

[0046] The drawings and the foregoing description illustrate examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined to form a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the actions of a flowchart need not be performed in the order shown; nor do all actions need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and use of materials, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0047] Advantages, further advantages, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, benefits, solutions to problems, and any components that may result in or enhance an advantage, benefit, or solution are not to be construed as critical, required, or essential features or components of any or all of the claims. REFERENCES 100 The system also includes a validation unit. 102 Bioinformatics Analysis Unit 104 Gene Construct Manufacturing Unit 106 Expression unit 108 cleaning unit 110 Folding unit 112 splitting unit 114 Characterization unit 116 Validation Unit

Claims

[1] A system for the production of soluble Trypanosoma Brucei gambiense phospholipase A2 (TbgPLA2) consisting of: a bioinformatics analysis unit configured to predict the 3D structure, topology, and signal peptide positions of TbgPLA2; a gene construct manufacturing unit configured to produce codon-optimized TbgPLA2 gene constructs with removed signal peptide and transmembrane sequences; an expression unit configured to express the TbgPLA2 constructs in host cells selected from the group consisting of E. coli and Pichia Pfarrer; a purification unit configured to purify TbgPLA2 from soluble fractions and inclusion bodies using nickel column metal affinity chromatography; and a refolding unit configured to renature TbgPLA2 using optimized buffer conditions consisting of Tris 55 mM, NaCl 21 mM, KCl 0.88 mM at pH 8.

2. [2] The system of claim 1, wherein the gene construct production unit is configured to produce constructs having N-terminal FLAG tag and C-terminal tags selected from the group consisting of 6xHis tag, EGFP tag and mClover tag. [3] The system of claim 1, wherein the expression unit is configured to express TbgPLA2 in E. coli cells selected from the group consisting of RIL, RIPL, Arctic Express, pLysS and Rosetta 2 cells. [4] The system of claim 1, wherein the purification unit is configured to solubilize inclusion bodies using 4 M urea and to purify both full-length TbgPLA2 at 58 kDa and truncated mature TbgPLA2 at 39 kDa. [5] The system according to claim 1, wherein the expression unit is configured to express TbgPLA2 in Pichia pastoris using the pPICZ vector with Zeocin resistance marker. [6] The system of claim 1, further comprising a cleavage unit configured to remove fusion tags by TEV cleavage and isolate free TbgPLA2 with a buffer containing 0.1% FOS-choline 12. [7] The system of claim 1, further comprising a characterization unit configured to determine enzymatic activity parameters, wherein the TbgPLA2 has a Vmax of 25,126 µmol / min and a Km of 1,194 mg / ml. [8] The system according to claim 1, wherein the gene construct manufacturing unit is configured to produce a codon-optimized TbgPLA 2- Gene with a codon adaptation index of 0.9 and a GC content of 57%. [9] The system of claim 1, further comprising a validation unit configured to verify TbgPLA2 expression by SDS-PAGE analysis, Western blotting with anti-His monoclonal antibody, and UV transillumination. [10] The system according to claim 1, wherein the system is configured to produce soluble TbgPLA2 with a specific activity of 107.14 µmol / min / mg for industrial applications selected from the group consisting of pharmaceutical applications, degumming agents, emulsifiers, detergent production and biodiesel production.

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