K. Bernaerts2, J. Impe2, K. Huygen3, J. Anné4 and L. Van Mellaert4

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K. Bernaerts2, J. Impe2, K. Huygen3, J. Anné4 and L. Van Mellaert4 Secretory production of biologically active Rv3804c antigen of Mycobacterium tuberculosis by Streptomyces lividans C. Vallin1*, E. Pimienta1, J.C. Ayala1, M. Sarzo1, C. Rodríguez1, I. Lule2, P.D. Huys2, K. Bernaerts2, J. Impe2, K. Huygen3, J. Anné4 and L. Van Mellaert4 1Institute of Pharmacy and Food, University of Havana, Havana, Cuba. 2Chemical and Biochemical Process Technology and Control Section, Department of Chemical Engineering, Katholieke Universiteit Leuven, Belgium, 3Immunology and Vaccinology, Scientific Institute of Public Health, Brussel, Belgium, 4 Bacteriology Department, Rega Institute, Katholieke Universiteit Leuven, Belgium *e-mail: val@infomed.sld.cu Tel: +53-7-8306484 1. Background M. tuberculosis Ag85A (Rv3804c), also known as Fibronectin- binding protein A, is an early secreted immunodominant antigen involved in cell wall mycoloylation. It represents one of the major secreted proteins from slowly growing mycobacteria. 2. Objectives 1. To evaluate the secretion of Ag85A-STII by S. lividans [pIJ-vsi-Ag85A-STII]1 grown in shaker and fermentor conditions. 2. To purify the antigens from S. lividans spent culture medium. 3. To evaluate the antigenicity of this protein. 3. Methods The detection and purification of the recombinant protein were achieved using AP-conjugated Strep-Tactin and Strep-Tactin Superflow® (IBA BioTAGnology), respectively. The fermentations were carried out in shaker flasks and bioreactors MBR 5 L (3.0 L working capacity, 400 rpm, 1 vvm, 27C) in TSB medium for 48 hours. Serological pattern of the antigen was studied by mean of an indirect ELISA using sera from human tuberculosis patients (80) and with sera from healthy individuals (controls) (33). 4. Results Ag85A-STII yield was at least 7 times higher when S. lividans [pIJ-vsi- Ag85A-STII] was grown in TSB medium in fermentor compared to shake flask condition Figure 1. Secretion of Ag85A-STII by S. lividans [pIJ-vsi-Ag85A-STII] grown in fermentor conditions. (A) Secretory yield of Ag85A-STII correlated with biomass of Streptomyces lividans TK24 [pIJ-vsi-ag85A-STII] grown in fermentor conditions. Ag85A-STII concentration was determined by means of slot blot (Life TechnologiesTM), Growth was estimated by measuring biomass dry weight (g/L). (B) Immunodetection of Ag85A-STII in extracellular fractions. The band amounts were estimated from densitometric scanning. 1, S. lividans [pIJ-vsi-Ag85A-STII] 40h, shake flask; 2, S. lividans [pIJ-vsi-Ag85A-STII] 48h, shake flask; 3, S. lividans [pIJ-vsi-Ag85A-STII] 44h, fermentor; 4, S. lividans [pIJ-vsi-Ag85A-STII] 46h, fermentor; 5, S. lividans [pIJ-vsi-Ag85A-STII] 48h, fermentor; 6, Ag85A. A B 1 2 3 4 5 6 KDa 45 30 23.5 Figure 2. Analysis of expression and purification of recombinant Ag85A-STII. (A) 12% SDS-PAGE stained with Coomassie blue R-250. 1- S. lividans TK24 [pIJ486] culture supernantant, 2- S. lividans TK24 [pIJ-vsi-rv3804c-STII] 24h, 3- S. lividans TK24 [pIJ-vsi-rv3804c-STII] 48h, 4- material precipitated with (NH4)2SO4 at 70% saturation cut, 5- Ag85A-STII eluted from the StrepTactin® column, 6- Precision Plus P Standard (IBA) (B) Amino acid sequence of the fusion region of preVsi-Ag85A and the N-terminal amino acid sequence of the recombinant Ag85A-STII. ↓: Signal peptidase cleavage site, Ag85A sequence in italics. Figure 3. Reactivity of sera from TB patients and healthy controls with recombinant Ag85A. (A) Scatter plot of individual seroreactivity against purified Ag85A analyzed by ELISA. Reactivity was tested with sera from human tuberculosis patients (80) and with sera from healthy individuals (controls) (33). The Mann-Whitney test was applied to determine the differences in mean optical densities (p < 0.05). Horizontal black line is the mean of the groups. (B) Sensitivity for Ag85A of a Cuban population of TB patients and negative TB sera. B KDa 37 25 1 2 3 4 5 6 A EAFSRPGLPV Ag85A band ..A Q A ↓ E A F S R P G L P V.. PreVsi-Ag85A (fusion region) N-terminal amino acid sequence Band A B TB patients (80) healthy controls (33) No. Positive Sensitivity No. Negative Specificity 59 73.75 % 25 75,76% 5. Conclusion Streptomyces lividans was a valuable host to produce a Mycobacterium tuberculosis protein with vaccine and diagnostic potential. Reference 1. Ayala JC, Van Mellaert L, Vallin C, Geukens N, Pimienta E, Rodríguez C, Sarzo M, González L, Anné J and Huygen K. 3rd Congress of European Microbiologists, Sweden 2009. Acknowledgements This work was partially supported by the Research Projects VLIR grant-ZEIN-2008PR346 of the "Vlaams interuniversitaire raad" (University Development Cooperation) in collaboration with the Rega Institute, Katholieke Universiteit Leuven, Belgium We thank Prof. Paul Proost (Rega Institute, K.U. Leuven) for N-terminal amino acid sequencing.