Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

Effect of temperature on the production of a recombinant antivenom in fed-batch mode.

In the pharmaceutical industry, nanobodies show promising properties for its application in serotherapy targeting the highly diffusible scorpion toxins. The production of recombinant nanobodies in Escherichia coli has been widely studied in shake flask cultures in rich medium. However, there are no upstream bioprocess studies of nanobody production in defined minimal medium and the effect of the induction temperature on the production kinetics. In this work, the effect of the temperature during the expression of the chimeric bispecific nanobody CH10-12 form, showing high scorpion antivenom potential, was studied in bioreactor cultures of E. coli. High biomass concentrations (25 g cdw/L) were achieved in fed-batch mode, and the expression of the CH10-12 nanobody was induced at temperatures 28, 29, 30, 33, and 37°C with a constant glucose feed. For the bispecific form NbF12-10, the induction was performed at 29°C. Biomass and carbon dioxide yields were reported for each culture phase, and the maintenance coefficient was obtained for each strain. Nanobody production in the CH10-12 strain was higher at low temperatures (lower than 30°C) and declined with the increase of the temperature. At 29°C, the CH10-12, NbF12-10, and WK6 strains were compared. Strains CH10-12 and NbF12-10 had a productivity of 0.052 and 0.021 mg/L/h of nanobody, respectively, after 13 h of induction. The specific productivity of the nanobodies was modeled as a function of the induction temperature and the specific growth rates. Experimental results confirm that low temperatures increase the productivity of the nanobody.Key points• Nanobodies with scorpion antivenom activity produced using two recombinant strains.• Nanobodies production was achieved in fed-batch cultures at different induction temperatures.• Low induction temperatures result in high volumetric productivities of the nanobody CH10-12.

A protocol for recombinant protein quantification by densitometry.

The protein purity is generally checked using SDS-PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS-PAGE: that is, imaged with Stain-Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10-12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS-PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10-12 was obtained through a fed-batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS-PAGE gel images.