Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

AIMS: The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase® cultivation mode and a redox mutant strain in the E. coli expression system. METHODS AND RESULTS: A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis® . CONCLUSIONS: This study demonstrated that the combination of SUMO fusion technology, EnBase® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. SIGNIFICANCE AND THE IMPACT OF THE STUDY: The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.

Novel materials for biofilm reactors and their characterization.

The application of adherently growing microorganisms for biotechnological production processes is established, but it is still a niche technology with only a small economic impact. However, novel approaches are under development for new types of biofilm reactors. In this context, increasingly more microstructured metal surfaces are being investigated, and they show positive effects on the bacterial growth and the biofilm establishment. However, for comparison of the data, the different surface materials have to correspond in their different characteristics, such as wettability and chemical composition. Also, new materials, such as plastic composite supports, were developed. To understand the interaction between these new materials and the biofilm-producing microorganisms, different surface science methods have to be applied to reveal a detailed knowledge of the surface characteristics. In conclusion, microstructured surfaces show a high potential for enhanced biofilm growth, probably accompanied by an enhanced productivity of the microorganisms.

Gene regulation of pteridine reductase 1 in leishmania promastigotes and amastigotes using a full-length antisense construct.

BACKGROUND: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. METHODS: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 µg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. RESULTS: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. CONCLUSION: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.