Team:Georgia State/Mambalgin

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Mambalgin

Background

Mambalgin is a potent analgesic polypeptide found in the venom of the Black Mamba snake (Dendroaspis polylepis). Mambalgin-1, the version found here, is a 3-finger toxin consisting of 57 amino acid residues forming 3 loops around a core in the shape of a hand. This protein has been found to inhibit acid-sensing ion channels (ASICs) in the central and peripheral nervous systems of mice through intraplanter and intrathecal injections. The inhibition of ASICs – important contributors to the pain pathway in both mice and humans – decreases sensitivity of nociceptive neurons to the perception of pain. The potency of mambalgin has been compared to the drug morphine. Unlike morphine, however, mambalgin has not shown an increase in tolerance over time (Diochot et al, 2012). Because of its potency and non-addictive properties, the potential of mambalgin as a pain reducer in humans is enticing. Synthetically producing this protein in large quantities would invalidate the need to harvest and extract directly from snakes – a dangerous and costly process.

Construct Design

Mambalgin E. Coli

For the design, we first codon optimized the Mambalgin-1 sequence (BBa_k1110003) for Escherichia coli using the IDT codon optimization tool. Restriction sites were then removed from the coding sequence that were iGEM incompatible. We added RFC 10 prefix and suffix and also included a start codon in the beginning of the sequence. Also included in the construct was a prokaryotic RBS (BBa_j61101), IPTG inducible promoter (BBa_R0011), and Myc and 6xHis epitopes. This fusion protein was designed specifically for easy expression in E. coli.

Experimental Design

Our experiment explores the possibility of synthetically producing the analgesic mambalgin-1 protein using E. coli and the yeast Pichia pastoris. Currently, the most effective form of extraction involves milking the venom from Dendroaspis polylepis (black mamba) – a dangerous and costly process. For E. coli, we inserted the mambalgin E. coli construct into the pSB1C3 vector to create a recombinant plasmid. This was then transformed into BL21 expression strain E. coli, which was IPTG induced in liquid cell culture.

Results

The 2015 GSU iGEM team was able to successfully express and characterize the mambalgin protein in E. coli. After inducing with IPTG overnight, the cell cultures were centrifuged and disrupted with a French press. Mambalgin was isolated using affinity chromatography, utilizing the 6x his tag in the construct. A SDS-PAGE was done, followed by a Ponceau S. stain to visualize the proteins. A band indicating a protein of ~9 kda – the size of the mambalgin for E. coli construct – was seen in the elute fraction.

Experimental Design

Mambalgin E. Coli

This entire section will be about mambalgin ecoli.This entire section will be about mambalgin ecoli.This entire section will be about mambalgin ecoli.This entire section will be about mambalgin ecoli.

Mambalgin Yeast

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Results

Mambalgin E. Coli

This entire section will be about mambalgin ecoli.This entire section will be about mambalgin ecoli.This entire section will be about mambalgin ecoli.This entire section will be about mambalgin ecoli.

Mambalgin Yeast

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