Search

Abstract Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.

Abstract Background Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. Results The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. Conclusion High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.

This work comprises three uneven parts. In the first part we have attempted to offer an abbreviated history of the operetta genre, from the beginning of the 17th century to its Hungarian composers at the end of the 19th and the beginning of the 20th centuries. Instead of an exhaustive historical presentation, we focused on pursuing a line of argumentation that would explain the emergence of the Hungarian composers from Budapest and Vienna, whose operettas successfully entertain audiences until this day. In the second part, we reviewed the specific topics approached by the Hungarian operetta composers, exemplifying these by briefly presenting the contents of the 14 operettas that we will analyze during our doctoral research. In the third part we compared the genres of opera and operetta, in terms of their elements. We considered this to be necessary in order to be able to demonstrate the specific features of the operetta genre and also emphasize the fact that they are very elaborate works created by talented and well-trained composers, and their staging required just as much talent, work and dedication throughout history, as their great operatic counterparts. [ABSTRACT FROM AUTHOR]

This hereby study has two main parts, uneven in length. In the first part we have attempted to classify the types of voices within the genre of operetta, while detailing their functional aspects, focusing on the analysis of the use of the soprano voice for the roles of prima donna and grande dame. In our second part, we have presented the main female roles of the 14 operettas that we will analyze within our doctoral research, classified from the standpoint of their authors. We have also added some interesting facts concerning the great Hungarian operetta composers, as well as mentioned famous performers who portrayed the main roles of these operettas during the time they were performed. [ABSTRACT FROM AUTHOR]

The increasing demand for recombinant antibodies as detection reagents in research, diagnostics, and therapy requires appropriate production systems. In contrast to antibody therapies, small recombinant antibody fragments like Fab and scFv are sufficient for most applications in research and diagnostics. These antibody fragments can also be produced in bacterial hosts. Gram-negative bacteria, particularly Escherichia coli, were extensively studied for the recombinant antibody production but they showed only a limited capacity to secrete antibody fragments into the medium--a prerequisite for easy downstream processing. Gram-positive bacteria are known to efficiently secrete recombinant proteins into the medium. Recently, we demonstrated the production of scFv and scFab fragments in Bacillus megaterium. Here, we describe the process in detail from transformation of B. megaterium to production and purification of scFv fragments.