4 - Heterologous Protein Expression

Question 1

List the issues with expression that can arise when the host is very phylogenetically different to the donor organism.

Answer 1

Loss of the gene or its expression.
Codon preference
mRNA instability

Question 2

Why are introduced genes often lost from their host genome?

Answer 2

Bacteria have very compact genome, and when forced to waste their resources on a recombinant protein they have a high selection pressure to excise or turn off expression of it.

Question 3

What is codon preference?

Answer 3

Many prokaryotes have a preference for the use of particular codons to avoid producing as many tRNAs. This means that eukaryotic genes are often not properly translated.

Question 4

Why do recombinant genes from eukaryotes often have issues with the stability of their mRNA?

Answer 4

The mRNA may require stabilising factors for expression in the eukaryote that are not present in the host. Bacterial hosts also sometimes produce factors to degrade the foreign mRNA.

Question 5

List the issues that may prevent recombinant proteins from taking on their natural wildtype structure.

Answer 5

Cofactor unavailability
Incorrect PTMs
Improper folding

Question 6

Why do eukaryotic proteins often misfold in prokaryotes?

Answer 6

Lack of specific molecular chaperones, reducing cytoplasm environment may lead to premature/incorrect disulphide formation.

Question 7

What is an expression system?

Answer 7

The sum of the different techniques employed to cause expression of the gene; the host species/strain and the plasmid promoter/copy number etc.

Question 8

What are the advantages of E. coli as an expression system?

Answer 8

Cheap and easy to grow/manipulate.
Well understood genetics.
Fast and easy to screen for most effective expression system.

Question 9

What are the disadvantages of E. coli as an expression system?

Answer 9

Unable to produce any PTMs, lack of folding systems.

Significantly different membranes, affects membrane protein folding and ligand binding.

Question 10

How suited to GPRC expression are E. coli?

Answer 10

Not very, many GPCRs require glycosylation and all are embedded in the membrane which is significantly different in bacteria.

Question 11

How are E. coli made more able to express GPCRs?

Answer 11

Protease deficient DH5a with a weak promoter used.
Long incubation at room temperature with high concentrations of their ligand.
Artificial signal sequences that target them to the periplasmic membrane.
Rare success from inclusion bodies, very difficult to refold.

Question 12

What are the advantages of yeast as an expression system?

Answer 12

Well understood genetics.
Easy transfection/screening for best expression system.
Capable of most PTMs and most eukaryotic folding facilities.

Question 13

How suited are yeast to GPCR expression?

Answer 13

Proper folding and PTM, and can be targeted to the plasma membrane, but mammalian GPCRs are often toxic to the cell so weak inducible promoter must be used.
Assortment of condition changes/ligand concentrations have been shown to improve yield.

Question 14

What are the advantages of insect cells as an expression system?

Answer 14

Eukaryotic, so almost identical PTMs and folding facilities, only sometimes differing in types of glycosylation.
Can be high yield.

Question 15

What are the disadvantages of insect cells as an expression system?

Answer 15

Some differences between insect and mammalian plasma membranes.
Labour intensive and time consuming. Not viable for large scale production.
Variability in PTMs mean proteins are rarely of crystal quality homogeneity.

Question 16

How well suited are insect cells to GPCR expression?

Answer 16

Expressed GPCRs tend to be fully viable. This is helped by the fact that insect cells do express some G proteins of their own.

Question 17

What are some examples of insect cells used as hosts?

Answer 17

Sf9 - Fall army worm cells (Spodoptera frugiperda)

Tni - Cabbage Looper (Trichoplusia ni)

Question 18

What are the advantages and disadvantages of mammalian cell expression?

Answer 18

Can be scaled up one a cell line has been established, but very low yield and highly complex to manipulate.

Question 19

What are the advantages of GPCR production in mammalian cells?

Answer 19

Totally correct folding and PTMs, cells express their own GPCRs so all co-receptors, antagonists and accessories already present and the membrane is the right composition.

Question 20

What are the disadvantages of GPCR production in mammalian cells?

Answer 20

Overexpression of the GPRCs is highly toxic to the cell, and working inducible systems are very difficult to design.

Question 21

What is cell free expression?

Answer 21

in vitro combination of all the components necessary for translation from the mRNA, to produce fully translated proteins sans the cells.

Question 22

What are the advantages of cell free expression systems?

Answer 22

Avoid issues of aggregation, toxicity, gene loss and host protein interference and allows for easy purification. Isotopic labelling is possible in this system as well as in vivo ones.

Question 23

What are the disadvantages of cell free expression systems?

Answer 23

Costly, labour intensive, impossible to scale up, no PTMs or folding systems.

Question 24

How is cell free expression suited to GPCR production?

Answer 24

Unfeasible, as not only does it lack folding and PTMs but also requires the tricky addition of membrane-mimetic liposomes.

Question 25

What is high throughput expression?

Answer 25

The automation of protein production and purification on a large scale, generally employing robotics technology.

Question 26

What field is high throughput protein production primarily useful in?

Answer 26

Pharmaceutical drug screening, where deriving structural and functional characteristics of a proteome allows for drug target identification.
Also used just to identify soluble proteins for potential study.

Question 27

What difficulties are caused by trying to purify much of the proteome of a single cell type?

Answer 27

Different expression levels, folding needs and biophysical properties make them difficult to all produce separately.

Question 28

How are the issues surrounding high throughput purification of a variety of proteins resolved?

Answer 28

Heterologous recombinant production allows for multiple proteins to be overexpressed and tagged in the same manner to allow for co-purification.

Question 29

What purification issues affect high-throughput techniques?

Answer 29

The same as those that cause problems on a smaller scale, aggregation and inclusion bodies etc. Fine tuning at each step can greatly improve yield of the process.

Question 30

How are high-throughput techniques used in systems biology?

Answer 30

To study the proteome as an integrated whole, high-throughput methods can be used to perform genome wide protein purification screens. Tandem affinity tage are used to ensure that complexes purify as a whole.

Question 31

How were high-throughput techniques used to study the yeast proteome?

Answer 31

6500 ORFs tagged
3000 expressible proteins found
2000 able to be purified

Question 32

What is synthetic biology?

Answer 32

A field concerned with engineering new biological systems or modifying and repurposing existing ones. High-throughput methods are used to supply raw materials for this.

Question 33

What is combinatorial domain hunting?

Answer 33

Shotgun fragment approach to heterologous expression to produce single domains of a protein for study, thus avoiding many of the folding issues that otherwise arise.

Question 34

How are high-throughput methods used in combinatorial domain hunting?

Answer 34

Shotgun approach to finding the domains requires them all to be expressed and purified to find those that fold properly.