PHASE 1 : Cloning of your gene of interest into a bacterial expression vector with His-tag or other selected tag (such as GST, His, or Thioredoxin):
- Amplification/isolation of the gene of interest out of a customer-supplied vector and subcloning it into a bacterial expression vector.
- Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.
- Transformation of recombinant constructs into a high efficiency expression bacterial strain.
- Mini-induction to over-express the target protein.
- Test for the expression of the recombinant protein by SDS-PAGE and/or western blot (customer must provide appropriate antibodies)if desired.
- Estimated time: 2-4 weeks.
PHASE 2 : Large-scale culture and purification:
- One litre of bacterial culture will be induced with IPTG and harvested for protein purification.
- Protein purification using Ni-NTA (for 6xHIS-tagged proteins) or glutathione (for GST-tagged proteins) beads at either native or denatured conditions.
- Solubility not guaranteed.
- Estimated time: 2 weeks.
5 μg construct, protein, documents (sequencing data,Western blot & SDS-PAGE result, design form).
If project stops after phase 1, products will include the above items minus protein.