Biotechnology L5

Front 1

What is the problem with E.Coli expression systems?

Back 1

They are not able to reproduce the range of post-translational modifications that are found in eukaryotes.
Recomb. proteins expressed in E.Coli can contain contaminant proteins which can cause immune response in host cell.
Eukaryotic proteins are often insoluble in E.Coli as correct chaperones are not present.

Front 2

Give 4 examples of post-translational modifications.

Back 2

1. Glycosylation- N-linked (sugar bound to asparagine) or O-linked (sugar bound to a serine or threonine)
2. Acetylation- acetate added
3. Palmitylation- addition of 16C chain to sulfhydryl group of cysteine
4. Phosphorylation- PO4- added

Front 3

What are post-translational modifications used for?

Back 3

Important for mode of action of therapeutics via target site.
Glycosylation could play an important role in folding and final conformation of the protein. It increases stability and prevents recognition of protein as foreign body.

Front 4

What is Saccharomyces cerevisiae?

Back 4

Model eukaryotic organism which can be grown easily and cheaply. It's genetically amenable like E.Coli.
It has 6000 genes and 2 plasmids, 2um plasmid most commonly used.

Front 5

What has been incorporated into the S.cerevisiae vector?

Back 5

Strong natural yeast promoters; glyceraldehyde phosphate dehydrogenase (GAPD), phosphoglycerate kinase, galactokinase and acid phosphatase.

Front 6

Why do we use shuttle vectors to produce recombinant proteins in S.cerevisiae?

Back 6

They allow for two different origins of replication so cloning can be done in E.Coli before transferring plasmids to saccharomyces

Front 7

What is the LEU2 selectable marker in S.cerevisiae?

Back 7

Fungi can't be killed by antibiotics so antibiotic resistance marker can't be used. Instead, we use mutants which can produce leucine in a growth medium which is negative for leucine. We know then that they have picked up the leucine gene and that of the recombinant protein.

Front 8

What features are there of a typical S.cerevisiae expression vector?

Back 8

Replication origin
LEU2- selectable marker for the plasmid in the yeast
GAPDp- promoter region
GAPDt- terminator region
cDNA- cDNA copy of gene
oriE- origin for replication in E.coli
Ampr- antiboiotic resistance gene

Front 9

How are proteins secreted by S.cerevisiae?

Back 9

1.Fuse a signal peptide from yeast matin type factor a1 gene
2. SRP targets signal peptide to translocon on ER membrane
3. Signal peptide is cleaved at Lys-Arg
4. Proteins are folded and modified in the ER lumen
5. A preassembled glycan is added to the Asn at Asn-X-Ser-Thr
6. Glycan is furthur processed in the golgi then secreted in a vesicle
7. Co-expression of a protein disulphide isomerase (like DsbA) leads to an increased yield of proteins containing disulfides.

Front 10

What are the problems with S.cerevisiae?

Back 10

-Often see plasmid loss from the population in fermenters, reducing yield
-Hyperglycosylation could occur. Where only 8-13 residues are usually added, an addition of 100 mannose residues in each N-linked side chin could alter the protein's properties, activity or immunogenicity.
-If a recombinant protein has a Asn-X-ser/Thr sequence expressed on its surface then it might be glycosylated when secreted even if it usually isn't.

Front 11

What is Pichia pastoris?

Back 11

A methylotrophic yeast which is very similar to S.cerevisiae but can give 10-100 fold greater levels of expression of intracellular and secreted proteins. It doesn't secrete many of its own proteins and doesn't hyperglycosylate proteins so is very good for secreted protein production.

Front 12

What naturally-occurring expression system is exploited in Pichia pastoris?

Back 12

Methanol is the sole carbon source for the enzyme alcohol oxidase which, when synthesised, makes up to 30% of soluble protein. You can use the alcohol oxidase promoter (AOX1p) and the termination and polyadenylation signal region (AOX1t) in expression vectors which allows you to control the expression of this enzyme. Repressed by glucose, induced by methanol.

Front 13

Give 3 examples of high yields of recombinant proteins from Pichia pastoris

Back 13

Tumour necrosis factor
Rat erythropoetin (EPO)
Alpha amylase

Front 14

How do you intgrate the Pichia pastoris vector onto the chromosome?

Back 14

1. Make a construct in E.coli; linearise and transform into Pichia for recombination
2. Integration occurs via homologous recombination between regions of the AOX gene. Select for His+ marker on the integrated DNA
3. For secretion, use yeast mating type factor a1 gene

Front 15

How is baculovirus used for protein expression in insect cells?

Back 15

First, clone the gene of interest into donor plasmid so that DNA is downstream of polyhedron promoter and followed by terminator sequences. Introduce it into E.coli containing a bacmid and helper plasmid. Recombination occurs between att regions.

Front 16

What is the bacmid?

Back 16

A genetically altered Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) which contains a E.coli origin, selectable marker and lacZ gene in the place of the polyhedron gene. We use it to prepare the recombinant baculovirus.

Front 17

How is plasmid uptake detected in the use of baculovirus?

Back 17

Insertion disrupts the lacZ on the bacmid so if the plasmid is taken up blue is shown (instead of white).

Front 18

How is the recombinant bacmid transfected into insect cells?

Back 18

It is purified from Ecoli and trnasfected into Spodoptera frugiperda, cell lines Sf9 or Sf21. These can't be used in continuous cultures because cells die at the end.

Front 19

How has baculovirus been modified?

Back 19

Co-expression of 2 genes at the same time using a second promoter called p10. This allows formation of complexes.
Some baculoviruses can infect some mammalian cell lines.

Front 20

What is the advantage of recombinant protein expression using insect cells?

Back 20

It is capable of many post-translational biotransformations
Can exploit the strength of the promoter used to make massive quantities of polyhedrin protein made later in the baculovirus infection cycle.

Front 21

What are the disadvantages of recombinant protein expression using insect cells?

Back 21

Cell lines aren't continuous
Cells have different gycosylation pathways to humans (although we can use different insect cells with similar modification pathways/ co-express human protein modification enzymes with recombinant protein.