Expression and characterization of a codon-optimized blood coagulation factor VIII.

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon-optimization of a B-domain deleted FVIII (BDD-FVIII). This resulted in 7-fold increase of the expression level in cell culture. The biochemical properties of codon-optimized BDD-FVIII were similar to the wild-type protein. SUMMARY: Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon-optimization of a B-domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD-FVIII variants expressed from codon-optimized and wild-type cDNAs (CO and WT, respectively). Methods Each variant of the BDD-FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7-fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had on average 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.

Characterizing a novel strain of Bacillus amyloliquefaciens BAC03 for potential biological control application.

AIMS: To identify and characterize a bacterial strain BAC03, evaluate its biological control activity against potato common scab (Streptomyces spp.) and characterize an antimicrobial substance produced by BAC03. METHODS AND RESULTS: Bacterial strain BAC03, isolated from potato common scab suppressive soil, was identified as Bacillus amyloliquefaciens by analysing sequences of fragments of the recA, recN, cheA and gyrA genes. BAC03 displayed an antagonistic activity against Streptomyces spp. on agar plates using a co-culture method. In glasshouse assays, BAC03 applied in potting mix significantly reduced common scab severity (P < 0·05) and potentially increased the growth of potato plants (P < 0·05). An antimicrobial substance extracted from BAC03 by ammonium sulfate precipitation was identified as an LCI protein using liquid chromatography-mass spectrometry. The antimicrobial activity of either a BAC03 liquid culture or the ammonium sulfate precipitate fraction was stable under a wide range of temperatures, and pH levels, as well as following incubation with several chemicals, but was reduced by all proteinases tested. CONCLUSIONS: Bacillus amyloliquefaciens strain BAC03 displayed a strong antimicrobial activity, that is, the suppression of potato common scab, and may potentially enhance the plant growth. LCI protein is associated with some of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strain BAC03 has the potential to be developed as a commercial biological control agent for potato common scab.