Mutagenesis and protein production Flashcards
(20 cards)
what are the flaws or risks with using mutagens
non specific
untargeted
hazardous
what is protein engineering and what properties can be changed
targeted modification of proteins by altering the coding sequence
requires knowledge of the structure and function of the protein
can be used to modify:
thermal stability
ph stability
kinetic properties
substrate specificity
what are some examples of engineered proteins
glucose xylose isomerase
petase
subtilisin
what is site directed mutagenesis
targeting of specific nucleotides within specific genes
what are the primary methods of site directed mutagenesis
m13 bacteriophage methods
pcr methods
synthetic gene method
how does m13 mutagenesis work
mutation induced using a mutated primer
converted to double stranded
m13 replicates as a ssdna
50 of new phages will have modification
plaques can be identified with hybridisation
what is pcr methylase modification
original dna is methylated with dam methylase
anneal mutant primers
amplify plasmid
digest original dna
put into mouth at eat it up
what is pcr splicing overlap extension
using two pcr reactions, one with a mutated forward, and the other a mutated reverse, results are then combined to overlap, new stand sythesised with overlap acting as a primer
what is artificial gene sythesis
multiple modified oligionucleootides, filled and ligated to make a highly modified strand
might use a kleinow fragment
what is scanning mutagenesis
systematically replacing codons to identify the purposes it serves
what is a common scanning mutagenesis substitution
alanine as it is low functional(like me fr)
what are the two requirements to produce a recombinant protein
a suitable host
an expression vector
what are the components of an expression vector
a promoter
ribosome binding site
terminator
may also contain a fusion tag to aids purification
what are some commonly used promoters
lac promoter
tac promoter
t7 promoter
how does the lac promoter function
lac1 binds and prevents the function, is displaced by the addition of lactose or iptg
derepression
what is the tac promoter
a hybrid promoter that is induced by iptg, and contains trp and lac regions
how does the t7 promoter
the t7 gene controls for the production of a t7 rna polymerase
this allows for the binding to a t7 site on a plasmid, this gene is controlled by a lac region
what is the arac and arabad promoters
arac binds to the arabad operon, unless arabinose is prescent in the medium
what is a fusion protein and what are some examples that may be used
a protein tag such as poly his tags
glutathione s transferase
maltose binding protein
this must be in frame
what are some issues that can occur when using ecoli
cannot excise introns
improper folding
improper processing
degredation of product protein
inoptimal codon bias
premature eja-termination
