Hands-On Learning in Recombinant Protein Research: A Study from Mahidol University’s Molecular Biosciences Program by Clarissa Elvaretta (012111133289)

 A scientific article by Clarissa Elvaretta, under the supervision of Dr. Cennikon Pakpahan, Sp. And., from the S1 Medicine program at Faculty of Medicine, Universitas Airlangga

From June 24 to July 19, I had the opportunity to participate in an outbound program at the Institute of Molecular Biosciences, Mahidol University, provided by Universitas Airlangga. During the first week, my peers and I attended workshops on micropipetting techniques, molecular cloning, vaccines, antibiotic susceptibility testing, and bacterial cytological profiling. In the subsequent weeks, we were divided into eight different laboratories. I chose to focus on molecular enzymology and cellular biology.

Molecular and cellular processes are broad and foundational to both basic and clinical research. Laboratories in this field use various approaches, including genomic sequencing, microscopy, and genetic models. In the molecular enzymology and cellular biology lab, I studied recombinant protein expression and purification, specifically focusing on the DNMT3A protein. Recombinant proteins are produced from genes cloned using recombinant DNA technology in heterologous expression systems. This process begins with identifying, isolating, and characterizing the desired gene, which is then inserted into vectors such as plasmids or bacteriophages using restriction enzymes and ligases. Protein expression involves creating an RNA copy of the DNA code (transcription) and translating that information into amino acid sequences to form proteins (translation). DNMT3A is a eukaryotic enzyme. In eukaryotes, transcription occurs in the nucleus, while translation happens in the cytoplasm. After translation, polypeptides are modified to complete their structure, determine their location, and regulate their activity within the cell.

Protein purification is crucial for analyzing individual proteins and their complexes and identifying interactions with other proteins, DNA, or RNA. The best purification protocol depends on the protein, the expression system used (e.g., prokaryotic vs. eukaryotic cells), and various other factors. Escherichia coli is commonly used for producing recombinant proteins due to its ease of use, rapid growth, and low culturing costs. Key steps in purification include charging, equilibration, binding, washing, and elution. DNMT3A, or DNA methyltransferase 3 alpha, is an enzyme that plays a critical role in regulating gene activity through DNA methylation. Its application in both biological and medical research is significant. In protein expression, DNMT3A is involved in regulating gene activity, while in protein purification, it focuses on isolating and studying the enzyme to understand its functions and applications.

Through this lab experience, I encountered various methods in protein expression and purification, including numerous challenges and errors due to incorrect techniques or lack of precision. However, each challenge provided valuable learning opportunities and helped improve my skills. This experience also reinforced the importance of ongoing evaluation and adaptation in scientific research. The knowledge and skills gained will be instrumental in addressing challenges in the medical field, enhancing research quality, and contributing to advancements in treatment and healthcare.

Overall, participating in the Molecular Biosciences Program at the Department of Molecular Enzymology and Cellular Biology provided an invaluable experience. The program allowed me to engage directly in experiments related to protein expression and purification, two critical aspects of molecular biology research. By mastering techniques such as gene cloning, protein expression, and purification, I not only refined my laboratory skills but also deepened my understanding of protein functions in cellular and biological contexts. This comprehensive experience has prepared me to contribute significantly to scientific research, drug development, and biotechnology applications, enriching my understanding of life sciences and equipping me for future challenges and opportunities in medical research and development.

References

medlineplus.gov. (n.d.). DNMT3A gene: MedlinePlus Genetics. [online] Available at: https://medlineplus.gov/genetics/gene/dnmt3a/.

Langlais, C. and Korn, B. (2020). Recombinant Protein Expression in Bacteria. Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine, [online] pp.1609–1616. doi:https://doi.org/10.1007/3-540-29623-9_4800.

‌www.sinobiological.com. (n.d.). Recombinant Protein Expression | Sino Biological. [online] Available at: https://www.sinobiological.com/resource/protein-review/protein-expression.

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