Introduction. Bacteria Is Used As Expression Host For Mammalian

1685 WordsMar 23, 20177 Pages
Introduction Bacteria is used as expression host for mammalian proteins due to its rapid and simple system of expressing recombinant proteins as it has a short doubling time. E.coli is a widely used system as there is more than enough knowledge about its ‘genome sequence, physiology and genetics’ (4). E.coli is a ‘gram-negative bacterium’ (5). Bacteria has a simple genetic manipulation with rapid expression which is suitable for large-scale fermentations and makes it easier to handle in a standard molecular biology laboratory (3). Also, it grows to ‘high densities which gives it a simple process scale-up’ and is less expensive due to its short generation time as bacteria can grow and multiply rapidly (4). It has ‘high level protein…show more content…
This reduces the soluble expression achieved. Also, ‘translation problems similar to those caused by codon usage bias can be created by high level expression of proteins having an abundant amino acid’ and also proteins might not be expressed in the bacteria host due to the overexpression in the cytoplasm as a lot of proteins form ‘inclusion bodies’ (5). Furthermore, ‘overexpression in bacteria can lead to differences in the codon usage which impedes translation’ due to the demand for one or more tRNAs that might be rare or lacking in the expression host. ‘Insufficient tRNA pools can lead to translational stalling, premature translation termination’ (5). (4) In expressing a mammalian protein into a bacterial host (4): • The transformation of competent bacteria cells is to be used to take up DNA sequence of interest. • The integration of the DNA into a bacterial genome or the circularization of the DNA sequence has to exist as a plasmid. • The transformed bacteria is selected using a selection marker which could be antibiotic. • The selected bacteria is expanded to a higher scale in an appropriate culture media for positive clones. • The plasmid is isolated or expressed as a vector. • The intracellular or secreted proteins of interest are then isolated and purified as transformants are scaled-up. Methodology for Practical 3 1. Make sure
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