Team:SDU-Denmark/Tour30
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P. A. S. T.
The difficulties with the bacterial production of peptide aptamers in E.coli comes from creating the variable part of the aptamer and in knowing that it matches a given target protein.
Two-hybrid screening is a way to determine protein-protein interactions or protein-DNA interactions. This system works by linking the proteins or DNA to each half of a transcription factor. The given transcription factor/or messenger protein is cut into 2 pieces. This leads to that the only way the transcription factor can relay a signal is of the 2 halves are connected, or close to each other. The system is designed so if the 2 proteins interact, this will bring together the 2 parts of the transcription factor/messenger protein and the signal will be relayed.
Our design is build around the catalytic activity of the adenylate cyclase in E.coli, which produces cAMP from ATP in the bacteria. The adenylate cyclase can be divided into 2 subunits, T18 and T25. When these 2 subunits come together it enables the production of cAMP. This is coupled together with a cAMP-induced promoter, which induces the production of Red Fluorescent Protein (RFP), which can be detected either by eye or by exposure to UV-light. The variable part of the peptide aptamer comes from a peptide library. This library consist different sequences generated from 20 random amino acids surrounded by a suffix and prefix, this can be considered the variable part of the peptide aptamer. This is inserted in a scaffold protein, Human Thioredoxin, which can be considered as the constant part of the peptide aptamer. If the amino acid sequence of the variable part matches the target protein, RFP will be expressed in the cells and we have created a peptide aptamer for the given target protein.
A process design has been made for large scale production of peptide aptamers. This is an 8 step process followed by purification of the peptide aptamers. It consists of 2 steps of autoclavation, 2 steps of fermentation and in the end an Expanded Bit Absorption (EBA). To put this into perspective a business plan was created to get an idea of what is nessesary to make a firm, which could produce peptide aptamers.