lec15W11

lec15W11 - Expressing eukaryotic proteins in bacteria: In...

Info iconThis preview shows pages 1–7. Sign up to view the full content.

View Full Document Right Arrow Icon
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
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
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?
Background image of page 2
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
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
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
Background image of page 4
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Reverse Genetics: Reverse genetic approaches are central to interpreting the information gained from genome sequencing efforts. Basically, it means you find a way to knock out or reducing the
Background image of page 6
Image of page 7
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 04/10/2011 for the course BIMM 101 taught by Professor Butler during the Spring '08 term at UCSD.

Page1 / 19

lec15W11 - Expressing eukaryotic proteins in bacteria: In...

This preview shows document pages 1 - 7. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online