enzyme engineering Flashcards
1
Q
protein biotechnology?
what do we use these proteins for?
A
- market for proteins = huge
1. use proteins for biologic drugs - insulin = 1st biotechnologically produced protein thats useful as a medicine
- growth hormones
- immunoglobulins
- anti-cancer drugs
2. laundry detergent - enzymes that breaj up stains - lipases
- amylases
3. fine chemicals
4. food processing - high fructose corn syrup using invatase
- amylase enzymes to process starch into sweet sugars.
2
Q
protein structure? (4 levels)
A
- proteins have evolved to suit the organisms they have evolved in
- we can engineer enzymes for better characteristics in our industrial processes
- we must understand how they work; function is related to structure
1. primary structure: - sequence of amino acids in a protein
- unique for each protein
2. secondary structure: - elements of hydrogen bonding giving structure to the protein
3. tertiary structure: - overall fold of the protein
4. quaternary structure: - proteins come together; either same type or different ones
3
Q
Primary protein structure
A
- 20 directly encoded amino acids
- selenocysteine
- linear arrangement of them (N-terminus to C-terminus)
- encoded in open reading frames in a genome/plasmid
- starts with start codon; ATG (methionine)
- ends in stop codon
4
Q
Secondary protein structure
A
- arrangement of primary structure into higher-level structures through hydrogen bonding in the backbone of amino acids
Alpha-helices: - Carbonyl (C=O) of one amino acid hydrogen bonded to amino group (N-H) of one of four amino acids down the chain
- turn ~3.6 amino acids
- side-chains stick out
- right handed (clockwise)
Beta strands and sheets: - hydrogen bonding between carbonyl and amino group across parts of primary structure
- antiparallel; planar hydrogen bonding, N-terminus or 1 strand adjacent to C-terminus of partner
- parallel; staggered hydrogen bonding, N- and C- termini of strands next to eachother
Disulfide bonds: - links between adjacent cysteine residues in oxidising conditions
- more common in extracellular proteins / eukaryotes
- stabilise structure
5
Q
disulfide bonds?
A
- links between adjacent cysteine residues in oxidising conditions
- proteins aren’t static, they move and wiggle, more so when higher temps
- we look at using disulfide bonds across the chain to make proteins more thermostable
- if you heat a protein to a high temp the secondary structure will go but it will be held in place by these disulfide bonds so that when temps lower the protein can fold again
6
Q
structural motifs?
A
- form super secondary structure
- the specific arrangement of multiple secondary structure elements together;
- greek key; antiparallel short loops
- BaB (beta-alpha-beta)
- aa (alpha-alpha)
7
Q
tertiary protein structure?
A
- overall fold of the protein chain
- stabilised by hydrogen bonding (secondary structure element)
- structural motifs
- disulfide bonds
- hydrophobic core; most proteins live in an aqueous environment so protein folds to get hydrophobic side chains away from water
- charged solvent exposed regions; my have hydrophobic patches interacting with other proteins (not ideal)
- integral membrane proteins have a hydrophobic ‘girdle’ (interacts with the membrane)
8
Q
quaternary protein structure?
A
- organisation of multiple protein chains together in a functional unit
- can be the same or different polypeptides
- stable (permanent) or transient (come together under certain signals)
- not all proteins do quaternary structure
- energetically favourable to form multimers of proteins
- eg. 60mer virus capsid
9
Q
enzyme active site structure?
A
- spatial organisation of catalytic residues and ligand binding pocket to give a protein its distinct function
tertiary/quaternary structure determines: - its catalytic activity
- its cofactor binding
- its substrate preference;
- enantioselective; preference for a
particular stereoisomer - Acyl chain length;
- Functional groups on ligands;
- enantioselective; preference for a
10
Q
bioprospecting for better enzymes?
A
- we want enzymes to do stuff that they don’t naturally
- we go to extreme environments
- microorganisms from hydrothermal vents
- screen bioinformatically for the enzyme of interest which we isolate
- antartic; enzymes active at low temps for laundry detergents
11
Q
protein engineering?
A
- how we can make the enzymes better, to do what we want
- site directed mutagenesis
- directed evolution (random mutagenesis)
12
Q
site directed mutagenesis: quickchange mutagenesis?
A
- codon insertions/deletions by mismatch PCR
- if we have a theory about the importance of a particular residue for catalysis we can swap it and observe the results
- works well for introducing a few mutations
- classic method = quickchange method from Agilent
- ideally make 2-3 changes as 1 will often revert back to wild type
1. plasmid with gene of interest
2. 2 primers that have a sequence mismatch to change 1 aa to another in the gene
3. plasmid is completely copied with mismatch = new mutated plasmid
4. get rid of parental DNA if plasmids are grown in a strain of E. coli that methylates the DNA
5. give it DP1 which cuts the methylated DNA
13
Q
site directed mutagenesis: mutation by inverse PCR?
A
- more complicated changes to protein structure
- fusions/deletions
- removal of residues/domains
- addition of peptide/protein tags
1. take plasmid with gene and design primers that introduce arbitrarily long insertions
2. primers are staggered from eachother producing a linear fragment of DNA (large substitutions/deletions can be achieved)
3. phosphorylate the ends back together
4. transform it
14
Q
directed evolution?
A
- used if you don’t know the structure of the protein (resquires a good assay)
- method that mimics natural selection with specific libraries of variant proteins
- selection for specific characteristics
- PCR-based methods = biased
1. take gene of interest in a plasmid
2. do mutagenic PCR - using a particular library of primers that are different / error prone polymerase that introduces point mutants
3. creates a library of mutants of your gene of interest
4. assay them; which ones give better characteristics in the assay
5. put the desired variants and sequence it
6. can repeat until desired characteristic is reached - Francis Arnold - makes proteins do things they don’t normally
15
Q
library generation in directed evolution?
A
- PCR-based methods; error-prone PCR (Taq/Mn2+) - error rate increased to 1/10 by swapping magnesium to manganese residue
- Primer-based methods; libraries of specific sequence variants
- random libraries; chemically synthesised libraries with large sequence variation
- mutagenic strains; lower DNA replication fidelity / chemical mutagenesis
