next-generation
TECHNOLOGies

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.
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In addition to our extensive library of pre-identified expression cassettes and the possibility of creating millions of unique expression vectors based on these libraries, we are developing a novel approach to expression vector design based on directed evolution.

With directed evolution we can create manyfold more unique expression vectors and thus overcome any potential limitations with pre-identified elements. We call this immensely powerful method VB Evolution.
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In each VB Evolution project we leverage our understanding of the Pm promoter architecture to generate billions of randomized expression vectors which are then screened in our proprietary screening platform that allows the rapid (1-2 weeks) screening of millions of expression vector variants. This HTP approach can be repeated in additional iterations to truly mimic nature's evolution and cause incremental improvements to the expression vectors.

The outcomes are newly evolved expression vectors that through evolution overcome any possible limitations from pre-identified elements. Thus we search the evolutionary space of hypothetical expression vectors to identify truly unique and optimal expression vectors for each and every protein.
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One of the main drawbacks of industrial production of proteins in E. coli is the lack of suitable secretion systems for transport of the proteins out of the cell.

This results in the protein accumulating internally, frequently resulting in the commonly observed issues of degradation, aggregation and misfolding, together with relatively high downstream costs as the proteins must be isolated and purified from the complex, internal environment, and possibly processed into biologically active forms.
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VB Secretion has been designed to overcome these issues by secreting the proteins into the more accommodating environment of the extracellular medium.

Central to VB Secretion is genetically modified E. coli strains that synthesize engineered flagella that function as channels for directed protein transport. Through specific tags, we can direct the transport of proteins out of the cell.

VB Secretion is particularly suitable for hard-to-express proteins that cause toxicity issues to the host cell, and for proteins where COGs are highly important and where the circumvention of downstream costs are important for a viable and economically feasible production process.

The VB Secretion technology is currently being finalized for E. coli.
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