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T. N. C. Ramya, Email: ramya@imtech.res.in
R.T.N.C. conceived the study. A.A. performed all experiments relating to metagenomic DNA isolation, phage display metagenomic library preparation, screening of the phage display library against all glycoconjugates, identification of enriched phage clones, cloning of MG1, MN3, MU1, and MU3 metagenomic inserts in pET-28a(+), and protein expression and purification of these recombinant proteins for glycan array analysis. M.L. performed biochemical characterization of MG1, MN3, MU1, and MU3, including enzyme linked lectin assays and all isothermal calorimetry experiments. S. Sunsunwal performed cloning into the expression vector for all metagenomic inserts other than MG1, MN3, MU1, and MU3, and protein expression and purification for all these clones other than St-Glc1 and St-Glc5/St-Glc5v2, and enzyme linked lectin assays for Lev-Lev5, StapPG-GlcNAc6, and PBGal-Gal8. A.Y. performed bioinformatics analysis of the metagenomic sequences. Kajal performed protein expression, purification, and enzyme linked lectin assays for St-Glc1 and St-Glc5v2. S. Subramanian performed the structure analysis of the metagenomic sequences. A.A. and R.T.N.C. wrote the first draft of the manuscript. All authors participated in experimental design, data analysis, and manuscript preparation and editing.
This work was supported by grants awarded by the Council of Scientific Research (CSIR) - BSC0119 (Twelfth Five Year Plan Network Project titled “Man as a super-organism: Understanding the Human Microbiome” awarded to TNCR and SS2) and OLP0178 (Research Council approved project titled “Exploring the function of novel microbial carbohydrate binding domains” awarded to TNCR). A.A. and M.L. acknowledge the Council of Scientific and Industrial Research (CSIR) for their fellowships, S. Sunsunwal and Kajal acknowledge the University Grants Commission for their fellowship, and A.Y. acknowledges the Department of Biotechnology (DBT), Government of India, for DBT-BINC-Junior Research fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors acknowledge Prof. F. William Studier, Brookhaven National Laboratory and Dr. Tatjana Heinrich, Institute for Child Health Research, Western Australia for the kind gifts of E. coli BL21 pAR3924,5453, E. coli BL24 pAR3924,5453, and the lysate of replication-deficient T7 Δ9-10B, D104, Δ38 phage, and the protocol for making T7 packaging extract, Mr. Zachary Klamer, Van Andel Institute, Michigan, for guidance in the use of MotifFinder for glycan array data analysis, the Protein-Glycan Interaction Resource of the CFG (supporting grant R24 GM098791) and the National Center for Functional Glycomics (NCFG) at Beth Israel Deaconess Medical Center, Harvard Medical School (supporting grant P41 GM103694) for the glycan array analysis, the CSIR-IMTECH mass spectrometry facility and the Taplin Mass Spectrometry Facility for mass spectrometry analysis, and CSIR-IMTECH (manuscript communication number 020/2022) for the research facilities and infrastructure.
The authors declare no competing interests.
© The Author(s) 2023