PREPARATION AND CHARACTERIZATION OF BOVINE MEAT AND MILK FACTOR VESICLES/PARTICLES & IDENTIFICATION OF MSBI1 REP INTERACTING PROTEINS

Sammanfattning: Introduction: Bovine meat and milk factors (BMMF) frequently isolated from bovine meat and milk, are discussed as putative causative agents for breast, colon and possibly other cancers. The hypothesis was formulated based on earlier correlative studies indicating that BMMFs could indirectly trigger inflammation, accumulate mutational events via reactive oxygen and nitrogen species production which eventually provokes tumor formation. Purpose: The study aimed to prepare and characterize BMMF particles or vesicles for future characterization and visualization, and identify possible interaction partners of BMMF1 Rep protein with human proteins from related carcinoma cell lines to better understand BMMF functions within the human host.  Methods: A plasmid-based overexpression of MSBI1.176 Rep C-HIS as representative of the intensively studied group of BMMF1 Reps was chosen for particle or vesicle preparations based on Cesium Chloride density gradient centrifugation. Co-immunoprecipitation experiments to detect the interaction of different BMMF1 and BMMF2 proteins were employed to identify possible helper or suppressor effects of different BMMF isolates and the correlated proteins which might be crucial for efficient production of BMMF particles/vesicles. Additional plasmids allowing codon-optimized Rep expression with specific tags were produced through cloning and verified by sequencing analysis.  Protein interaction assays were conducted to analyze possible interaction partners of the MBSI1.176 Rep within the human host by pull-down assay based on five different cell lines: monocytes, MCF-7, RKO, Colo678, and HEK293TT cells. Results: Particle/vesicle preparation experiments indicated that although MSBI1 specific DNA and protein were detected in fractions of high-molecular-weight, no evidence was found that the desired protein is initiating a specific formation of capsomeres or vesicles. Two new plasmids (for overexpression of N- or C-terminally Flag-tagged codon-optimized MSBI1 Rep) were designed and successfully cloned within the study.  The newly designed cloned plasmids were suitable for Immunoprecipitation (IP) and Co-immunoprecipitation (Co-IP) experiments indicating the interaction of the MSBI1.176 Rep proteins with each other as a hint for the formation of Rep dimers, oligomers or aggregates. IP/ Co-IP results were optimized by modification of different lysis and washing buffer conditions allowing conduction of future experiments using combinations proteins of isolates to analyze the interaction of proteins of the BMMF1 and BMMF2 group. Importantly, protein interaction experiments based on MSBI1.176 Rep pulldown followed by mass spectrometry for verification of putative Rep interaction partners identified the two proteins RFWD2 and CEP83 as putative Rep interaction partners within all five cell systems tested. The current result indicates the interaction of MSBI1 protein with host proteins, which might have a role in cancer induction. More detailed experiments will have to follow to more specifically determine the BMMF functions crucial for formulation and understanding of preventive strategies of BMMF infection.