Class notes

Structural Biology- Protein Expression and Engineering

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Course
Biochemistry (LIFE50022)

Institution
Imperial College London (ICL)

Lecture notes on the topic Protein Expression and Engineering, taught by Professor Bernadette Byrne at Imperial College London. Course: Bsc Biochemistry. Module: Structural Biology.

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Uploaded on February 1, 2023
Number of pages 6
Written in 2022/2023
Type Class notes
Professor(s) Bernadette byrne
Contains All classes

biochemistry
structural biology
protein expression
protein engineering
imperial college london

Institution
Imperial College London (ICL)
Education Unknown
Course
Biochemistry (LIFE50022)

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TITLE DATE

Protein Engineering
↳
modifying protein sequence by substitution or indels

Purposes -

probe mechanism (generate specific substitution, observe effect on structure & function (
-create novel proteins (w/ new functions and ..
applications (
e.g. improve catalytic function of enzyme
-alter substrate specificity or stereospecificity
-improve stability (stable enough for downstream applications (
-biotech applications 4: requirements 4
↳
e.g. more eco-friendly production

Mutagenesis
synthetic gene route: design substitution in specific locations

e.g. sub out of CyS
send to a
company like Thermo Fisher

time efficient
*

plasmid-based approach
e.g. Quikchange protocol from Stratagene (company (

1.
identify target site for mutation in parental

Plasmid DNA

2. high temperature to denature plasmid
lower temperature to anneal mutagenic
oligonucleotide primers

3.
Amplify DNA using PCR so new strand contains

mutation
add Dpn l enzyme

4. Wild
type (parental) DNA has restriction sites

by DpnI
cleares wild type, all remaining DNA is mutated

5. Transform mutated plasmid (nicked, dsDNA) into
XL1- Blue supercompetent cells and then E. coli

6. Sequencing repair nicks produce more
mutated DNA
↳ check for correct mutation, avoid introduction of random mutations
https://bynikkib.com

, TITLE DATE

Mutate genomic DNA (non-plasmid): overlap extension method using PCR

4 Oligonucleotide primers:

-forward and reverse primers

Introduces mutation

Mutagenesis (overlapping region
3: -upstream and downstream
5'
primers contain specific
5' 31
restriction enzyme sites

produce 2 PCR products
-

Oligo 104 (upstream + reverse
-

oligo 203(downstream+ forward)

denature and anneal in presence of

-full length pck product w/ both unique RE sites at oligo primers 102

3' and 5' ends and mutation at the middle

DNA polymerase makes as DNA

cut wild type and mutagenic DNA at 2 RE sites, replace WT w/ mutated and anneal

Alanine Scanning Mutagenesis
↳
systematic approach to engineer proteins (change a
region / whole protein one AA at a time
-used to investigate role of residues
-
obtain a more stable construct

mutate every amino acid residue into Ala one by one

Ala
*

is mutated into Lea

means of assessing the effect of mutations relative to wild type: normally Functional Assay

example: used to stability of G-protein coupled receptor (serrano-vega etal, 2008 (

systematic ... slow

https://bynikkib.com

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