METHODS: rPkMSP-119 protein was heterologously expressed using Expresso® Solubility and Expression Screening System and competent E. cloni® 10G cells according to protocol. Three rounds of biopanning were performed on purified rPkMSP-119 to identify binding peptides towards rPkMSP-119 using Ph.D.™-12 random phage display library. Binding sites of the identified peptides to PkMSP-119 were in silico predicted using the CABS-dock web server.
RESULTS: Four phage peptide variants that bound to PkMSP-119 were identified after three rounds of biopanning, namely Pkd1, Pkd2, Pkd3 and Pkd4. The sequences of both Pkd1 and Pkd2 consist of a large number of histidine residues. Pkd1 showed positive binding signal with 6.1× vs. BSA control. Docking results showed that Pkd1 and Pkd2 were ideal binding peptides for PkMSP-119 .
CONCLUSION: We identified two novel binding peptides of PkMSP-119 , Pkd1 (HFPFHHHKLRAH) and Pkd2 (HPMHMLHKRQHG), through phage display. They provide a valuable starting point for the development of novel therapeutics.
METHODS: Colon tissues (normal and cancerous) were homogenized and the proteins were extracted using three protein extraction buffers. The extraction buffers were used in an orderly sequence of increasing extraction strength for proteins with hydrophobic properties. The protein extracts were separated using the SDS-PAGE method and the images were captured and analyzed using Quantity One software. The target protein bands were subjected to in-gel digestion with trypsin and finally analyzed using an ESI-ion trap mass spectrometer.
RESULTS: A total of 50 differentially expressed proteins in colonic cancerous and normal tissues were identified.
CONCLUSION: Many of the identified proteins have been reported to be involved in the progression of similar or other types of cancers. However, some of the identified proteins have not been reported before. In addition, a number of hypothetical proteins were also identified.
METHODS AND RESULTS: Transmission electron microscopy revealed phage pPM_01 to be a siphovirus (the first reported virus to infect P. mirabilis), with its complete genome sequence successfully determined. The genome was sequenced using Illumina technology and the reads obtained were assembled using CLC Genomic Workbench v.7.0.3. The whole genome contains a total of 58,546 bp of linear double-stranded DNA with a G+C content of 46.9%. Seventy putative genes were identified and annotated using various bioinformatics tools including RAST, Geneious v.R7, National Center for Biotechnology Information (NCBI) BLAST, and tRNAscan-SE-v1.3 Search. Functional clusters of related potential genes were defined (structural, lytic, packaging, replication, modification, and modulatory). The whole genome sequence showed a low similarity to known phages (i.e., Enterobacter phage Enc34 and Enterobacteria phage Chi). Host range determination and SDS-PAGE analysis were also performed.
CONCLUSIONS: The inability to lysogenize a host, the absence of endotoxin genes in the annotated genome, and the lytic behavior suggest phage pPM_01 as a possible safe biological candidate to control P. mirabilis infection.
MATERIALS AND METHODS: Human and caprine MFGM proteins were isolated and analyzed, initially by polyacrylamide gel electrophoresis, and subsequently by quadrupole time-of-flight liquid chromatography-mass spectrometry. This was then followed by database search and gene ontology analysis. In general, this method selectively analyzed the abundantly expressed proteins in milk MFGM.
RESULTS: Human MFGM contains relatively more abundant bioactive proteins compared with caprine. While a total of 128 abundant proteins were detected in the human MFGM, only 42 were found in that of the caprine. Seven of the bioactive proteins were apparently found to coexist in both human and caprine MFGM.
RESULTS/DISCUSSION: Among the commonly detected MFGM proteins, lactotransferrin, beta-casein, lipoprotein lipase, fatty acid synthase, and butyrophilin subfamily 1 member A1 were highly expressed in human MFGM. On the other hand, alpha-S1-casein and EGF factor 8 protein, which are also nutritionally beneficial, were found in abundance in caprine MFGM. The large number of human MFGM abundant proteins that were generally lacking in caprine appeared to mainly support human metabolic and developmental processes.
CONCLUSION: Our data demonstrated superiority of human MFGM by having more than one hundred nutritionally beneficial and abundantly expressed proteins, which are clearly lacking in caprine MFGM. The minor similarity in the abundantly expressed bioactive proteins in caprine MFGM, which was detected further, suggests that it is still nutritionally beneficial, and therefore should be included when caprine milk-based formula is used as an alternative.