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MBY364 Theme 9 summary
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Full summary of theme 9 (expression in E. coli) made using lecture notes, the textbook and class notes.
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EXPRESSION IN E. COLIRECOMBINANT PROTEIN PRODUCTION IN E. COLI
- Synthesis of a functional protein depends on
- Transcription of the gene - promoter, transcriptional terminator
- Translation of the mRNA - ribosome binding site, SD sequence
- Post-translational processing of the protein
- The promoter activity must be low in order to be controlled - an expression
vector can aid in this
- Problems may arise at different levels of protein production
- Transcriptional level
- Foreign gene may contain sequences that act as termination signals in
E. coli
- Innocuous in normal host cell but in E. coli it will result in premature
termination and a loss of gene expression
- Between transcription and translation
- E. coli is unable to excise introns from primary mRNA transcripts
- Have to use cDNA clones derived from mRNA which lacks introns
- Translation level
- Although the genetic code is almost universal, there may be bias
towards individual codons
- A codon bias is reflected by the abundance of the relevant tRNAs with
a relative lack of the tRNAs for the less frequently used codons
- If a foreign gene contains a high proportion of unfavoured codons,
translation may be hindered by the low abundance of tRNA needed to
decode it
- After translation
- Degradation of protein by host proteases - use a mutant E. coli host
strain (e.g. OmpT-; lon-) which is deficient in proteases responsible for
the protein degradation
- E. coli does not perform post-translational modifications (e.g.
phosphorylation, glycosylation) on heterologous expressed proteins
- At the level of the cell
- High expression levels might be harmful to the host
- The energy drain causes by overexpression of cloned genes may
disadvantage the recombinant E. coli in terms of growth and cell
stability when compared to non-recombinant E. coli
- Bacteria whose plasmids have been altered/lost can outgrow the
recombinants, resulting in loss of product formation
- Expression of foreign proteins can be achieved by
- Cloning of a DNA cassette containing all the elements needed for
transcription and translation
, - Cloning of the gene in a specially designed expression vector
EXPRESSION VECTORS
- Enable a cloned gene to be placed under the control of a promoter that
functions in E. coli
- Are required if one wants to prepare RNA probes from the cloned gene or to
purify large amounts of the gene product
- Many of these vectors can be regulated easily by changes in the growth
conditions of the host cell
- E. coli RNA polymerase
- Is a multisubunit enzyme
- Core enzyme consists of two identical ⍺ subunits and one
each of β and β’ subunits
- Core enzyme is not active until the σ factor is present
- Recognises different types of promoters depending on which type of σ
factor is attached - most common is σ70
- Optimal promoter:
- -35 region - TTGACA
- -10 region - TATAAT
- RNA pol must bind to both sequences to initiate transcription
- Promoter strength is directly proportional to the degree of similarity with
the consensus sequence
- Spacer region is important - must be exactly 17bp
- More or less than 17bp results in a weaker promoter
- Upstream elements (UP) consist of AT-rich sequences that
increase transcription by interacting with RNA pol ⍺ subunit
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