Phase 1. Design and synthesis of a gene of interested protein
Searching the library for the gene of interested protein.
Codon optimization of the gene to match that of the expression system, thus to boost the protein expression level.
Synthesis of the gene.
Time: 1~2 weeks
Phase 2. Cloning of the target gene into a bacterial expression vector
Amplification and isolation of the gene and subcloning it into a bacterial expression vector.
Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.
Maxi-prep of the recombinant vector DNA.
Time: 1 week
Phase 3. Protein expression and purification
Transformation of recombinant constructs into high-efficiency expression bacterial strain.
Selection of bacterial colonies for protein expression.
2 liters of bacterial culture will be induced with IPTG and the cell pellets will be harvested for protein purification.
Cell pellets disruption by sonication.
Protein purification by a variety of columns including affinity, gel filtration, ion exchange and hydrophobic column.
Remove the unwanted fusion part or purification tag by a protease, and purify the required protein.
Provide 5~10 mg target protein at native or denatured conditions. The purity of the protein is above 80% .
Test for the result of each step by SDS-PAGE or Western blot (if desired, and customer must provide appropriate antibodies).
Note: The native condition just means that the protein solution buffer does not contain any denaturant, such as urea and guanidinium hydrochloride. We do not guarantee the activity of purified protein since we can not test it.
Time: 2~3 weeks
Phase 4. Refolding and endotoxin removing of target protein
Using up to 18 kinds of renaturing buffer and several methods to optimize the refolding condition.
Removing the endotoxin of target protein, make it ready to be used in cell culture assays or preclinical studies.
Time: 2 weeks
