Key to Vectron's technologies is the highly tunable bacterial promoter Pm and its cognate transcription regulator gene xylS. Pm is an inducible promoter that is tight when uninduced and very strong under maximal induction.
Pm is induced with low doses of benzoic acids which are low-cost and non-toxic to both bacteria and humans and does not exhibit the usual "all-or-nothing" effect as seen with IPTG induced promoters.
Through engineering of Pm and xylS, and Pm’s 5'-UTR region, novel expression cassettes have been obtained that can rival the T7 promoter in expression strength yet still retain their [RF1] tunable nature.
We use Pm/xylS in conjunction with minimal replicons of the RK2 plasmid which gives very low metabolic burden, highly stable vectors in the absence of antibiotic selection pressure, and the possibility of increased vector copy number for increased titers. These expression vectors can be used in any E. coli strain.
Obtaining high titers of soluble protein in E. coli can be challenging, especially for human proteins at grams per liter scale. In our experience this is usually not solved by one single approach but through incremental improvements resulting from the careful selection and engineering of different expression elements. Central to this are the following:
- The Pm/xylS system allows finely tuned expression which can be key to achieving soluble expression of many proteins, especially when inclusion bodies are formed because the capacity of the folding machinery is the bottleneck. This is in contrast to most other E. coli expression systems that overburden the folding machinery resulting in large amounts of inclusion bodies.
- Translocation to the periplasm for more efficient disulfide bond formation. Vectron has a library of signal peptides that can be used for efficient translocation of different proteins.
- Finally, the company has extensive experience with improving solubility through the use of solubilization partners (fusion proteins), chaperons, and cultivation adjustments.
Up to 60 g/L
Proven technology that has been validated through record-high titers and is now used for marketed products.