Vectron is a well-rounded expression technology and services provider. In addition to the core proprietary technologies listed below, the company is highly experienced in the use of other bacterial expression systems and technologies.
Pm/xyls for controlled gene expression in E. coli
At the centre of Vectron's technologies is the highly controllable promoter Pm and its cognate transcription regulator xylS. Pm is an inducible promoter that is tight when uninduced and very strong under maximal induction. It is induced with 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 their 5'-UTR region, novel expression cassettes have been obtained that can rival T7 in expression strength yet still retain their controllable nature. The Pm/xylS expression cassette can be used for controlled expression of proteins in various bacteria, at very high levels, but also for more controlled expression ideal for metabolic engineering. Pm/xylS is usually used 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 titres, but can also be used on other replicons. In contrast to T7, Pm/xylS does not require specific E. coli strains, and can, to our knowledge, be used for recombinant expression in any E. coli strain.
This technology is covered by the following patent applications: USPTO6258565, EPO922108, USPTO12/376312 , EPO2059599, USPTO12/520139, EPO2078076.
Obtaining high titres of soluble protein in E. coli can be challenging, especially for human proteins at grams per litre scale (which is the case for most biopharmaceutical APIs). In our experience this is usually not solved by one single method but through incremental improvements resulting from adoption of various technologies and methods. Central to this are the following:
- The Pm/xylS system allows finely tuned expression which can be key to achiving 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.
- Vectron is currently developing VB Solutions, a new technology that can be used in connection with Pm/xylS to improve solubility of many proteins. VB Solutions does not require any changes to the protein sequence but may turn a predominantely insoluble protein into a predominantly soluble protein.
- 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.
Alternative bacterial hosts
E. coli is the preferred host for bacterial production of recombinant proteins, especially biopharmaceutical APIs. For other applications alternative bacterial hosts can be advantageous to circumvent weaknesses of E. coli, including low titres for some proteins, low secretion, inclusion body formation, etc. Vectron's core technologies have a broad host-range and can be used for controlled expression of many proteins in other bacteria. We are currently developing a toolbox of alternative hosts for production of challenging proteins.
dualUTR for increased transcriptional and translational control
Optimizing translational efficiency by engineering the 5'-UTR without negativelly affecting transcriptional efficiency can be challenging and in many cases impossible. Vectron filed in 2015 a patent application protecting a method of designing synthetic 5' UTR sequences that allows the engineering of a 5'-UTR sequence for both optimal transcription and translation at the same time.