First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin.

Anthochlor pigments (chalcones and aurones) play an important role in yellow flower colourization, the formation of UV-honey guides and show numerous health benefits. The B-ring hydroxylation of chalcones is performed by membrane bound cytochrome P450 enzymes. It was assumed that usual flavonoid 3'-hydroxlases (F3'Hs) are responsible for the 3,4- dihydroxy pattern of chalcones, however, we previously showed that a specialized F3'H, namely chalcone 3-hydroxylase (CH3H), is necessary for the hydroxylation of chalcones.

Molecular and Enzymatic Characterization of Flavonoid 3'-Hydroxylase of × .

× (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2'--glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic × plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3'-hydroxylase (F3'H), which catalyzes B-ring hydroxylation of flavonoids.

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles.

Biopharmaceutical drug substances are generally produced using fermentation technology and are subsequently purified in the following downstream process. For the determination of critical quality attributes (CQAs), such as target protein titer and purity, monitoring tools are required before quality control analysis. We herein present a novel reversed phase liquid chromatography method (RPLC), which enables facile and robust protein quantification during upstream and downstream processing of intracellularly produced proteins in E.

The Effects of Lactose Induction on a Plasmid-Free T7 Expression System.

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions.

Recombinant production of a hard-to-express membrane-bound cytochrome P450 in different yeasts-Comparison of physiology and productivity.

Cytochrome P450s comprise one of the largest protein superfamilies. They occur in every kingdom of life and catalyse a variety of essential reactions. Their production is of utmost interest regarding biotransformation and structure-function elucidation.

E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3).

Background: Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG.

Recombinant production of eukaryotic cytochrome P450s in microbial cell factories.

Cytochrome P450s (P450s) comprise one of the largest known protein families. They occur in every kingdom of life and catalyze essential reactions, such as carbon source assimilation, synthesis of hormones and secondary metabolites, or degradation of xenobiotics. Due to their outstanding ability of specifically hydroxylating complex hydrocarbons, there is a great demand to use these enzymes for biocatalysis, including applications at an industrial scale.

Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains.

When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (q) and the corresponding maximum specific lactose uptake rate (q) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv).