BackgroundThe production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein.ResultsIt was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation–refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer.ConclusionsMaltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1053-9) contains supplementary material, which is available to authorized users.

Dengue being one of the deadliest diseases of tropical regions, enforces to put continuous efforts for the development of vaccine and effective therapeutics. Most of the antibodies generated during dengue infection are non-neutralizing and cause antibody dependent enhancement. Hence, making a potent neutralizing antibody against all four dengue serotypes could be very effective for the treatment. However, designing a single antibody for all serotypes is difficult due to variation in protein sequences. Therefore, the objective is to identify conserved region of dengue envelope protein and then develop an antibody against that conserved region. Before advancing to the development of such an antibody, it is desirable to validate the interactions between antibody and dengue envelope protein. In silico analysis of such interactions provides a good platform to find out a suitable region to design and construct an antibody against it by analyzing antigen-antibody interaction before synthesizing the antibody. In this study, two highly conserved regions of dengue envelope protein were identified and an scFv was constructed against it. Both scFv and FuBc proteins were expressed in bacterial expression system and binding efficiency was analyzed by SPR analysis with KD value 2.3 μM. In order to improve binding efficiency, an in silico scFv mutant library was created which was virtually screened for higher binding efficiency. Six mutants with high binding efficiency were selected for further analysis. The binding ability of these mutants were predicted using simulation analysis which shows these mutations were stabilizing scFv-FuBc complex.

This chapter describes the major ex situ and core collections (including landraces, wild relatives, elite germplasm and cultivars), aiming to explore the genetic diversity of the global germplasm collections; in situ conservation; characterization and evaluation of collections for key agronomic traits (such as disease resistance, yield and quality); and utilization of germplasm of lentil.

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Dengue virus (DENV) is a vector-borne human pathogen that usually causes dengue fever; however, sometime it leads to deadly complications such as dengue with warning signs (DWS+) and severe dengue (SD). Several studies have shown that fusion (Fu) and bc loop of DENV envelope domain II are highly conserved and consist some of the most dominant antigenic epitopes. Therefore, in this study, Fu and bc loops were joined together to develop a short recombinant protein as an alternative of whole DENVenvelope protein, and its immunogenic potential as fusion peptide was estimated. For de novo designing of the antigen, Fu and bc peptides were linked with an optimised linker so that the three dimensional conformation was maintained as it is in DENV envelope protein. The redesigned Fubc protein was expressed in E. coli and purified. Subsequently, structural integrity of the purified protein was verified by CD spectroscopy. To characterise immune responses against recombinant Fubc protein, BALB/c mice were subcutaneously injected with emulsified antigen preparation. It was observed by ELISA that Fubc fusion protein elicited higher serum IgG antibody response either in the presence or in absence of Freund's adjuvant in comparison to the immune response of Fu and bc peptides separately. Furthermore, the binding of Fubc protein with mice antisera was validated by SPR analysis. These results suggest that Fu and bc epitope-based recombinant fusion protein could be a potential candidate towards the development of the effective subunit vaccine against DENV.