Expression of L-Amino Acid Oxidase (Ml-LAAO) from the Venom of the Micrurus lemniscatus Snake in a Mammalian Cell System
Abstract
1. Introduction
2. Results and Discussion
2.1. Optimization and In Silico Analysis of ML-LAAOnat and ML-LAAOrec
2.2. Expression Optimization
2.3. Reverse Transcription Followed by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
2.4. Evaluation of Ml-LAAOrec Enzymatic Activity
2.5. Purification and Identification of Ml-LAAOrec
2.6. Mass Spectrometry
3. Conclusions
4. Materials and Methods
4.1. Obtaining the Amino Acid Sequence of Ml-LAAO
4.2. In Silico Analysis
4.3. Ml-LAAO Expression
4.3.1. Plasmid Construction and Amplification
4.3.2. Cell Culture and Transfection
4.3.3. Optimization and Efficiency Assessment
4.4. RNA Extraction and Quantitative Reverse Transcription PCR (qRT-PCR)
4.5. Determination of Ml-LAAOrec Activity
4.6. Ml-LAAOrec Purification and Quantification
4.7. SDS-PAGE and Western Blot Assay
4.8. Sample Preparation (In-Solution Digestion) for Nano Liquid Chromatography (Nano-LC) Coupled to Electrospray Ionization Spectrometry (ESI)
4.9. Search for Identities
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
| LAAO | L-amino acid oxidase |
| Ml-LAAO | L-amino acid oxidase from Micrurus lemniscatus |
| Ml-LAAOnat | Native Ml-LAAO |
| Ml-LAAOrec | Recombinant Ml-LAAO |
| DMEM | Dulbecco’s Modified Eagle’s Medium |
| EGFP | Green Fluorescent Protein |
| PBS | Phosphate-buffered saline |
| SDS-PAGE | Sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
| HPLC | High-performance liquid chromatography |
| DDBJ | DNA Data Bank of Japan |
| qRT-PCR | Quantitative reverse transcription polymerase chain reaction |
| RNAse P | Ribonuclease P (endogenous control) |
| FBS | Fetal bovine serum |
| OPD | o-Phenylenediamine |
| BSA | Bovine serum albumin |
| TFA | Trifluoroacetic acid |
| TCEP | Tris(2-carboxyethyl)phosphine |
| NTC | No template control |
| ESI | Electrospray ionization |
| LC-MS/MS | Liquid chromatography tandem mass spectrometry |
| MS/MS | Tandem mass spectrometry |
| PBS-T | PBS with Tween-20 |
| H2O2 | Hydrogen peroxide |
| HCD | Higher-energy collisional dissociation |
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| Properties | Ml-LAAOnat | Ml-LAAOrec |
|---|---|---|
| Number of nucleotides | 1503 | 1629 |
| Number of amino acids | 501 | 543 |
| Molecular weight | ~57 kDa | ~62 kDa |
| Theoretical Isoelectric Point | 9.02 | 8.97 |
| Half-life estimate (in vitro, in mammalian cells) | 30 h | 30 h |
| Instability index | 33.17 (stable) | 35.55 (stable) |
| Aliphatic index | 83.71 | 83.54 |
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© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
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Costa, A.J.d.O.; Matavel, A.; Campos, P.C.; Santos, J.L.d.; Ataide, A.C.Z.; Leclercq, S.Y.; de Alvarenga, V.G.; Caldas, S.; Castro-Borges, W.; Borges, M.H. Expression of L-Amino Acid Oxidase (Ml-LAAO) from the Venom of the Micrurus lemniscatus Snake in a Mammalian Cell System. Toxins 2025, 17, 491. https://doi.org/10.3390/toxins17100491
Costa AJdO, Matavel A, Campos PC, Santos JLd, Ataide ACZ, Leclercq SY, de Alvarenga VG, Caldas S, Castro-Borges W, Borges MH. Expression of L-Amino Acid Oxidase (Ml-LAAO) from the Venom of the Micrurus lemniscatus Snake in a Mammalian Cell System. Toxins. 2025; 17(10):491. https://doi.org/10.3390/toxins17100491
Chicago/Turabian StyleCosta, Ari Junio de Oliveira, Alessandra Matavel, Patricia Cota Campos, Jaqueline Leal dos Santos, Ana Caroline Zampiroli Ataide, Sophie Yvette Leclercq, Valéria Gonçalves de Alvarenga, Sergio Caldas, William Castro-Borges, and Márcia Helena Borges. 2025. "Expression of L-Amino Acid Oxidase (Ml-LAAO) from the Venom of the Micrurus lemniscatus Snake in a Mammalian Cell System" Toxins 17, no. 10: 491. https://doi.org/10.3390/toxins17100491
APA StyleCosta, A. J. d. O., Matavel, A., Campos, P. C., Santos, J. L. d., Ataide, A. C. Z., Leclercq, S. Y., de Alvarenga, V. G., Caldas, S., Castro-Borges, W., & Borges, M. H. (2025). Expression of L-Amino Acid Oxidase (Ml-LAAO) from the Venom of the Micrurus lemniscatus Snake in a Mammalian Cell System. Toxins, 17(10), 491. https://doi.org/10.3390/toxins17100491

