Protein engineering Flashcards Preview

BIO2030 Biotechnology > Protein engineering > Flashcards

Flashcards in Protein engineering Deck (13)
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1
Q

What is a protein’s secondary structure?

A

Higher-level structures formed from backbone interactions between amino acids

2
Q

Describe α helices

A

Carbonyl of one amino acid and the amino group of an amino acid 4 down the chain hydrogen bond
Turn every ~3.6 amino acids
Side chains stick out
Right-handed

3
Q

Describe β strands/sheets

A

Hydrogen bonding between carbonyl and amino group across parts of the primary structure
Can be antiparallel (planar H bonding and N- and C- termini are adjacent to partner) or parallel (Staggered H bonding where N- and C- termini is adjacent)

4
Q

Describe disulfide bonds

A

Links between adjacent cysteine residues in oxidising conditions
More common in extracellular proteins and eukaryotes
Stabilise structures

5
Q

What are structural motifs?

A

Arrangements of multiple secondary structure elements together

6
Q

What are active sites?

A

Spatial organisation of catalytic residues and ligand binding pockets to give a protein its function

7
Q

What are fusions/deletions?

A

Removal of residues and domains or the addition of tags

8
Q

What is site-directed mutagenesis?

A

Codon indels by mismatch PCR

9
Q

What is directed evolution?

A

Mimics natural evolutionary selection with specific libraries and selection for specific protein characteristics

10
Q

What are the pros of directed evolution?

A

Doesn’t require detailed structural knowledge of protein of interest

11
Q

What are the cons of directed evolution?

A

PCR based methods can be biased
Selection method required
Needs high-throughput assay that measures what you want to evolve

12
Q

What are some ways to generate a library?

A

PCR-based methods
Primer-based methods
Random libraries (buy)
Mutagenic strains

13
Q

What are some ways to make a protein more stable or enhance its activity?

A
Thermostability 
Salt tolerance 
Solvent tolerance 
pH tolerance 
Greater substrate range 
Altered cofactor preference
Enantioselectivity
New chemistry