Protein expression customization is an important service in the field of biotechnology, which involves the process of expressing a foreign gene to a target protein in a host cell. This process consists of multiple steps, from gene cloning to protein purification, and requires careful design and operation. The following is an overview of a typical custom protein expression process:
1.Gene cloning.
First, the gene of the protein of interest needs to be cloned into an appropriate expression vector. This usually involves the following steps:
PCR amplification: The gene sequence of interest is amplified from the source DNA using PCR technology.
Digestion and ligation: DNA is cleaved using restriction enzymes and attached to the expression vector.
Transformation: The constructed recombinant plasmid is introduced into host cells, usually E. coli or mammalian cells.
2.Expression optimization.
Once the gene of interest has been successfully cloned into the expression vector, expression conditions need to be optimized to ensure efficient expression of the protein of interest. This may involve the following:
Promoter selection: Select the appropriate promoter to regulate the transcription level of the gene.
Signal peptides: If an extracellular secretory protein is required, the appropriate signal peptide sequence needs to be added.
Temperature and growth conditions: Optimize culture conditions, including temperature, pH, and media composition, to maximize expression of the protein of interest.
3.Protein expression.
Once the expression conditions have been optimized, protein expression can be performed
Inducible expression: Inducers (such as IPTG or isopropylthiogalactoside) are added at the appropriate growth stage to induce expression of the gene of interest.
Culture: Continue culturing cells under expression conditions to promote protein expression.
4.Protein purification.
The final step is to purify the expressed protein of interest from the cell
Cell disruption: Proteins are released using mechanical methods or by dissolving the cell membrane.
Purification: A variety of separation techniques such as affinity chromatography, ion exchange chromatography, gel filtration, etc., are used to purify the protein of interest.
Concentration: Concentrate the purified protein solution to the desired concentration.
5.Validation and analysis.
Finally, the purified protein needs to be validated and analyzed to ensure its quality and functionality
SDS-PAGE and Western Blotting: Used to measure the molecular weight and purity of proteins.
Mass spectrometry: Determine the amino acid sequence and modifications of proteins.
Biological Function Analysis: Tests the biological activity of proteins.
Conclusion. The custom protein expression workflow is a complex and delicate process that requires systematic experimental design and operation. Through rational design and rigorous operation, it is possible to achieve efficient expression and purification of proteins with specific functions, providing important support for research in the field of biomedicine and biopharmaceuticals.