Enzyme expression improvement platform
Enzyme expression improvement platform
Engineering of E. coli expression platform resulted in improved soluble protein production
A) SDS-PAGE showing serial dilution of baseline and enhanced protein expression.
B) Graphical representation of fold improvement of Almac custom enhanced expression relative to baseline expression.
One of the key cost contributors for a biocatalytic process is the enzyme. If enzyme loading can be reduced, this reduces the economic burden and makes the process more economically feasible. Almac have enhanced their in-house E. coli protein expression by modifying the expression platform. These modifications have been shown to produce on average a 4-fold improvement in soluble expression of proteins of interest when compared with the original, baseline expression. This telescopes into the generation of higher yields in fed batch and large-scale fermentations for the same volume of E. coli culture. Almac has proven that this system works for multiple enzyme classes.
Yeast expression of an animal derived lipase
A. SDS-PAGE showing soluble secreted protein expression. Host: WT strain (as negative control); Lip2KO: empty knock out control (to ensure no endogenous lipases have been upregulated with the knock out); Lipase: Desired lipase containing strain.
B. Activity assay results showing increased hydrolysis observed with enzyme expressed in Yeast compared to enzyme expressed by E. coli in an equal volume of culture.
Yeast expression of an animal derived lipase
A. SDS-PAGE showing soluble secreted protein expression. Host: WT strain (as negative control); Lip2KO: empty knock out control (to ensure no endogenous lipases have been upregulated with the knock out); Lipase: Desired lipase containing strain.
B. Activity assay results showing increased hydrolysis observed with enzyme expressed in Yeast compared to enzyme expressed by E. coli in an equal volume of culture.
Almac’s eukaryotic expression system
For enzymes that need posttranslational modifications or need to be produced in strains that are naturally endotoxin free and generally considered as safe (GRAS) an alternative expression platform can be beneficial. Within INSIGHT, Almac have developed a modular synthetic toolbox to generate an eukaryotic secretory, high yielding, non-lipopolysaccaride producing GRAS organism.
This organism is suited to the expression of enzymes or proteins which are not suitable for E. coli expression. With interchangeable genetic elements, promoters, integration sites and selection markers this system is extendable to future improvements and multifunctional designs.
As a proof of concept, animal derived lipases were inserted into this expression system and using optimised growth conditions better expression and higher activity levels were demonstrated than that obtained by standard E. coli expression systems.
Publications
Examples of where Almac enzyme engineering technology has been used to improve an enzyme