RAD51D Human

RAD51D is a crucial protein involved in the homologous recombination repair pathway, which is essential for maintaining genomic stability by repairing DNA double-strand breaks. This protein is one of the five RAD51 paralogs in humans, the others being RAD51B, RAD51C, XRCC2, and XRCC3 . These paralogs work together to facilitate the repair of DNA damage, thereby preventing chromosomal abnormalities and maintaining genomic integrity .

RAD51D plays a significant role in the homologous recombination repair mechanism. It is involved in the formation of RAD51 nucleoprotein filaments on single-stranded DNA, which is a critical step in the homologous pairing and strand exchange process . This process is vital for the accurate repair of DNA double-strand breaks, which can otherwise lead to genomic instability and cancer .

Mutations in the RAD51D gene have been associated with an increased risk of ovarian and breast cancers . These mutations impair the protein’s ability to repair DNA, leading to an accumulation of genetic damage and an increased likelihood of cancer development .

The preparation of human recombinant RAD51D involves the use of various expression systems, including bacterial, yeast, and mammalian cell lines. The choice of expression system depends on the desired yield, post-translational modifications, and functional activity of the recombinant protein .

1. Bacterial Expression Systems: These are commonly used due to their simplicity and high yield. However, they may lack the necessary post-translational modifications required for full functionality of the protein.
2. Yeast Expression Systems: These provide a balance between yield and post-translational modifications. Yeast cells can perform some eukaryotic post-translational modifications, making them suitable for producing functional recombinant proteins.
3. Mammalian Cell Lines: These are used when the recombinant protein requires complex post-translational modifications. Although they have lower yields compared to bacterial systems, they produce proteins that are more similar to their natural counterparts in terms of structure and function .

The synthetic routes for producing recombinant RAD51D involve cloning the RAD51D gene into an appropriate expression vector, transforming the vector into the chosen host cells, and inducing protein expression . The expressed protein is then purified using various chromatographic techniques to obtain a high-purity product .

1. Gene Cloning: The RAD51D gene is amplified using polymerase chain reaction (PCR) and inserted into an expression vector.
2. Transformation: The expression vector is introduced into the host cells (bacteria, yeast, or mammalian cells) through transformation or transfection methods.
3. Protein Expression: The host cells are cultured under conditions that induce the expression of the recombinant RAD51D protein.
4. Protein Purification: The expressed protein is purified using techniques such as affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography to achieve high purity .

The reaction conditions for the expression and purification of recombinant RAD51D vary depending on the expression system used. Key factors include temperature, pH, and the presence of specific inducers or supplements in the culture medium .

1. Temperature: Optimal temperature for protein expression varies with the host system. For bacterial systems, it is typically around 37°C, while for yeast and mammalian systems, it ranges from 28°C to 37°C.
2. pH: The pH of the culture medium is maintained within a specific range to ensure optimal growth and protein expression. This range varies with the host system.
3. Inducers and Supplements: Specific inducers (e.g., IPTG for bacterial systems) and supplements (e.g., amino acids, vitamins) are added to the culture medium to enhance protein expression and stability .

The industrial production of recombinant RAD51D involves scaling up the laboratory procedures to large-scale bioreactors. This process includes optimizing the culture conditions, scaling up the fermentation process, and implementing stringent quality control measures to ensure the consistency and purity of the final product .

1. Scale-Up: The laboratory-scale culture conditions are optimized and scaled up to large bioreactors, which can hold several liters to thousands of liters of culture medium.
2. Fermentation: The host cells are cultured in the bioreactors under controlled conditions to maximize protein yield. Parameters such as temperature, pH, and oxygen levels are closely monitored and adjusted as needed.
3. Purification: The recombinant protein is purified from the culture medium using large-scale chromatographic techniques. The purification process is designed to remove impurities and ensure the high purity of the final product .
4. Quality Control: Stringent quality control measures are implemented to ensure the consistency, purity, and functionality of the recombinant protein. This includes testing for contaminants, verifying the protein’s structure and function, and ensuring compliance with regulatory standards .