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lec15W11 - Expressing eukaryotic proteins in bacteria In...

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Expressing eukaryotic proteins in bacteria: In cloning the luxAB genes into E.coli, we were able to use a genomic sequence to get protein expression Because eukaryotic genes have introns, you cannot usually clone eukaryotic genomic DNA and get protein expression in bacteria (only DNA replication for sequencing) Instead, mRNA sequences are reverse transcribed into DNA sequences (cDNA) and the cDNA is cloned into a plasmid for expression Although there can be problems expressing eukaryotic proteins in bacteria, it is often done successfully, and bacteria are usually the first choice since they are easy and cheap to grow
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First, transcription of sequence Eukaryotic cDNA will not contain a bacterial promoter or terminator These must be present in vector upstream and downstream of polylinker in order for transcription to occur Second, translation of mRNA Eukaryotic sequence will definitely not have RBS, so this must be present in vector, downstream of promoter Eukaryotic sequence may not be full length, so may not have start or stop codon – so these must also be present in vector. All these signals surround the polylinker region, since cDNA will be cloned here What’s needed for expression of a eukaryotic protein in bacteria?
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Therefore, the vector used for cloning and expressing a eukaryotic protein in bacteria must have a bacterial promoter followed by an RBS with start codon just after the RBS – all of these signals are before the polylinker region. The vector will usually also have a stop codon and and a terminator signal after the polylinker region. promoter Polylinker region RBS start codon stop codon terminator
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Problems with expressing proteins in bacteria: Protein is toxic to bacteria Protein needs to be post-translationally modified E. coli has trouble translating the proline codons in human genes Examples of human proteins being made via recombinant technology Insulin – E.coli Factor Vlll –cultured mammalian cells Human growth hormone – E.coli or yeast FSH – yeast Interferon – E.coli
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Reverse Genetics: Reverse genetic approaches are central to interpreting the information gained from genome sequencing efforts.
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