Recombinant protein production involves several key steps, each crucial for the successful expression and purification of the target protein. Here is an overview of the typical process:

1)  Gene Cloning:

Gene Identification: Identify and isolate the gene encoding the target protein.

Vector Construction: Insert the gene into a suitable expression vector (plasmid), which will carry the gene into the host cell.

2)  Transformation:

Host Cell Selection: Choose an appropriate host cell for protein expression (e.g., E. coli, yeast, mammalian cells, insect cells).

Introduction of Vector: Introduce the recombinant vector into the host cells through a process called transformation (for bacteria and yeast) or transfection (for mammalian and insect cells).

3)  Selection and Screening:

Selection: Grow the transformed cells on selective media to ensure that only cells that have taken up the vector survive.

Screening: Identify the cells that contain the recombinant vector with the correct insert and orientation.

4)  Expression:

Induction: Induce the expression of the target protein using specific conditions (e.g., adding an inducer like IPTG in bacteria).

Optimization: Optimize the expression conditions (temperature, medium composition, induction time) to maximize protein yield.

5)  Cell Harvesting:

Cell Growth: Grow the host cells to a sufficient density.

Harvesting: Collect the cells by centrifugation or filtration after the expression phase.

6)  Protein Purification:

Cell Lysis: Lyse the harvested cells to release the recombinant protein. This can be done using physical methods (e.g., sonication, French press) or chemical methods (e.g., detergents, enzymatic lysis).

Initial Purification: Use techniques like centrifugation or filtration to remove cell debris and obtain the crude protein extract.

Chromatography: Purify the target protein using chromatographic methods (e.g., affinity chromatography, ion exchange chromatography, size exclusion chromatography). Affinity tags (e.g., His-tag, GST-tag) often aid in this process.

Polishing: Further purify the protein to remove any remaining impurities and achieve the desired purity level.

7)  Quality Control and Analysis:

Verification: Verify the identity and purity of the recombinant protein using techniques like SDS-PAGE, Western blotting, and mass spectrometry.

Activity Assay: Assess the functionality of the purified protein through appropriate biochemical assays.

8)  Storage:

Stabilization: Add stabilizing agents (e.g., glycerol, protease inhibitors) to maintain protein stability.

Storage Conditions: Store the purified protein under conditions that preserve its activity and integrity (e.g., -80°C for long-term storage).

Each step in the process is critical and may require optimization depending on the specific protein and host system used. This workflow ensures that the recombinant protein is produced efficiently, with high purity and activity.

GenCefe provide one-stop solutions from codon optimization, gene synthesis, to protein expression and purification. In addition to four conventional expression systems of bacterial, yeast, insect cell, and mammalian cell, we have also developed a patented cell free expression technology, which is suitable for the production of difficult proteins such as toxic proteins and membrane proteins.