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Chimeric Construction and Gene Expression of Butelase (B1) and Viola Uliginosa Plant (Vu)A

Undergraduate #55
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Shanice Fezeu - Benedict College
Co-Author(s): Michael Summers, Clemson University, Clemson, SC



Macrocyclic peptides are generally more resistant to proteolysis and often have higher potency than linear peptides and so they are excellent leads in drug design. Their study is significant because they offer potential as a new generation of drugs that are potent, specific, and have fewer side effects than traditional small molecule drugs. Viola Uliginosa is a plant from the violaceae family, represented by (Vu). The violaceae family is rich in species renowned for producing specific head to tail cyclic peptides known as cyclotides with numerous biological activities. Butelase 1 is the first asparagine (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 (B1) cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s-1 and catalytic efficiencies as high as 542,000 M-1s-1, butelase 1 is the fastest peptide ligase known. Previous studies have shown that Butelase 1 could provide a complementary approach for peptide macrocyclization which would result in unique structural and biopharmaceutical properties. Therefore, it was hypothesized that chimerized constructs of Vu, Vum, and B1 would produce an active and efficient cyclized protein product with the stability of Vu and activity of B1. Three Vu and B1 digestion constructs were joined by ligation which were Vu+B1, B1+Vu, and B1+Vum. With transformation, the Vu, Vum, and B1 genes that encode protein ligases were inserted in the plasmid of E. Coli cells via electroporation. IPTG was added, which binds to the plasmid to activate the gene for protein expression. Maldi Tof Activity Assay was used to detect ligated activity. The samples tested were cell lysate and a purified sample using His-Tag Beads. Both types of samples were run with either reaction A (4.5ul enzyme & 0.5ul substrate) or reaction B (1ul enzyme, 0.5ul substrate & 3.5ul pH6buffer). Out all the three constructs, only B1+Vum and Vu+B1 cell lysate under reaction A produced ligated product in the activity assay, which included cell lysate as the enzyme. Ligation concentration was higher after the overnight time point. All the reaction samples under reaction B didn’t produce cyclized activity. It has been proven that cell lysate conditions are more efficient in this study. Western blot was used to confirm the proper cyclized product. Future research will investigate constructs of different amino acids and point mutations of Butelase 1 will be tested for increased stability.
References: Nguyen,K.T., Wang, S., Qiu.Y., Hemu, X., Lian.Y., & Tam, P.L. 2014. Butelase 1 is an ASX-Specific Ligase enabling peptide macrocyclization and synthesis. Nature Chemical Biology. 10:732-738. I:10.1038/NCHEMBIO.1586
.

Craik,D.J., & White,A.M. 2016. Discovery and Optimization of Peptide Macrocycles. Expert Opinion on Drug Discovery. 11:12, 1151-1163. DOI:10.1080/17460441.2016.1245720

Not Submitted

Funder Acknowledgement(s): I would like to acknowledge Dr. Huang Mei as the Research Mentor at Nanyang Technological University in Singapore. Funding was provided by NSF/HBCU-UP Grant to Dr. Samir Raychoudhury. Grant No. HRD 1436222; SC LS-AMP Award # 1305050

Faculty Advisor: Dr. Huang Mei, huangmei@ntu.edu.sg

Role: PCR using Q5 High-Fidelity DNA Polymerase, Gel Electrophoresis using Argarose Gel, Centrifugation Purification (EZNA Cycle Pure Kit), DNA Digestion, Centrifugation Purification, Ligation, Transformation of Top10 Competent Cells w/ Ligation Products via Electroporation, Cell Culture w/ Antibiotic Resistant Plate, Plasmid Mini Prep Spin Protocol, Transformation w/ T7 Shuffle Cells via Heat Shock, Protein Induction, Sonication, Protein Purification Using His-Tag Beads, Activity Assay using MALDI TOF

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This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

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