Synthesis of a New Scytovirin Protein Derivative, Sd1-Sd2-Sd2, to Decrease Hiv-1 Binding Affinity

Wood, Alexandra M (2013) Synthesis of a New Scytovirin Protein Derivative, Sd1-Sd2-Sd2, to Decrease Hiv-1 Binding Affinity. Undergraduate thesis, under the direction of John Rimoldi from Medicinal Chemistry, University of Mississippi.

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Scytovirin is a novel anti-viral protein isolated from the cyanobacterium Scytonema varium. This protein has produced great scientific interest for its anti-human immunodeficiency virus (HIV) activity. Scytovirin disrupts the mechanism of HV infection by binding to HIV-1 proteins glycoprotein 120, glycoprotein 160, and glycoprotein 41. It does not bind to the CD4 receptor on human immune cells. The wild-type protein consists of a single 95 amino acid chain. When folded, Scytovirin’s amino acid sequence is arranged into two distinct domains, each with a high degree of sequence conservation. This implicates the importance of both domains in anti-HIV activity. Primers were designed and constructed to synthesize three novel, mutant, forms of the Scytovirin protein in an attempt to increase the anti-HIV activity of Scytovirin. Each gene analog featured coding to increase the size of the new mutant proteins by the addition of the second domain onto the nearly wild-type protein. The amino acid sequence connecting this domain was unique to each mutant. Single amino acids were replaced at numerous locations in each domain through site-directed mutagenesis to facilitate proper folding of the proteins. Once the gene constructs were sequenced and purified, all three mutants were expressed in Escheria coli, each with thrombin (THR) fused to its N-terminus. The protein constructs, three versions of SD1-SD2-SD2, were cleaved from THR with restriction enterokinase (rEK). Soluble protein was purified using polyhistidine tag affinity purification through FPLC, and HPLC. NMR determined the structures of the proteins and if proper folding had occurred. SD1-SD2-SD2 wild-type and short linkers were expressed in Origami cells, purified, and examined by 2D Heteronuclear NOESY NMR. NMR of SD1-SD2-SD2 wild-type linker indicated that the protein had not folded completely. Additional research involving the expression and purification parameters of SD1-SD2-SD2 is required to obtain proper folding before SD1-SD2-SD2 will be screened against HIV-1 to assess anti-viral activity.

Item Type: Thesis (Undergraduate)
Additional Information: Research was performed at the University of Alabama in Huntsville
Creators: Wood, Alexandra M
Student's Degree Program(s): BA Biology
Thesis Advisor: John Rimoldi
Thesis Advisor's Department: Medicinal Chemistry
Institution: University of Mississippi
Subjects: Q Science > QD Chemistry
R Medicine > RM Therapeutics. Pharmacology
Depositing User: Alexandra M Wood
Date Deposited: 06 May 2014 18:57
Last Modified: 06 May 2014 18:57

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