lecture 20 - recombinant proteins Flashcards
What are the main steps in the prokaryotic production of recombinant proteins?
Isolate gene of interest via PCR, clone into expression plasmid, transform plasmid into bacteria for expression, grow cells expressing protein of interest, isolate and purify the protein
What is cDNA?
complementary DNA - DNA synthesised from a SSRNA template in a reaction catalysed by the enzyme reverse transcriptase.
Why is cDNA useful for recombinant protein production?
It contains no introns, so can be processed by prokaryotic cells
Why is recombinant insulin produced by making the A and B chains separately?
Natural insulin is produced in the pancreas as a pre-protein that is processed by the golgi. This process cannot occur in bacterial cells so the chains must be made separately and joined later on to form functional insulin
What gene is used in plasmids to form fusion proteins with Insulin A and B chains in recombinant insulin production?
The Lac Z gene
What is a Lac Z/ insulin fusion protein?
A protein that contains either an A or B insulin subunit as well as protein coded for by the Iac Z. This is the prerequisite required to manufacture functioning insulin, and is made in E. coli prokaryotic hosts.
What bonds must form between the A and B chains to create active insulin?
Disulfide bonds
What type of cells are usually used in the production of recombinant insulin?
Prokaryotic cells - usually E. coli.
What are the advantages of prokaryotic production of recombinant proteins?
Low cost, high yield, pathogen free
What are the disadvantages of prokaryotic production of recombinant proteins?
Proteins often partially folded, unable to perform post-translational modifications
What are the steps in eukaryotic production of recombinant proteins?
Isolate cDNA, clone into eukaryotic expression plasmid, transform plasmid into bacteria to replicate, transfect plasmid into eukaryotic cells, extract recombinant protein from cell media, purify protein
What is the difference between transformation and transfection?
Transformation is the transfer of foreign DNA into a bacterial, plant or yeast cell, while transfection is the introduction of foreign DNA to a eukaryotic mammalian cell
Why are eukaryotic cells useful for processing of recombinant proteins?
They have Endoplasmic Reticulum, golgi, etc. that allow for natural processing
What are the advantages of mammalian/eukaryotic cell production of recombinant proteins, such as insulin?
Cells have organelles (e.g. ER and golgi) that will naturally process complex proteins. The proteins are also excreted from the cells, making for easier processing.
What are the disadvantages of mammalian/eukaryotic cell production of recombinant proteins, such as insulin?
Expensive
