1. Chapter 7 Heterologous Protein Production in Eukaryotic Cells

2. Eukaryotic Protein Produced in Prokaryotic Expression System
Unstable
Biologically inactive
Improper protein folding
Eukaryotic system has protein disulfide isomerase (PDI) for correct disulfide bond formation
Lacking proper posttranslational modification
Proteolytic cleavage of a precursor form
Glycosyltaion : 30% of mammalian proteins
O-linked : Ser, Thr (-OH) (Fig. 7-1)
N-linked : Asn (-CONH2) (Fig. 7-2)
Phosphorylation, acetylation, sulfation, acylation, g-carboxylation
Addition of fatty acids
Myristoylation (myristylation) C14
Palmitoylation (palmytylation) C16
Contamination with toxic compounds (pyrogens)

3. O-linked Glycosylation
Yeasts
Insects
Mammals

4. N-linked Glycosylation
Yeasts
All start with the same initial group, which is subsequently trimmed and then elaborated in diverse ways within and among species.
C. Mammals
B. Insects

5. Eukaryotic Expression Vector
Component of shuttle vectors
Eukaryotic promoter and terminator
Markers (both for E. coli and eukaryotes)
Replication origins for E. coli and eukaryotes (optional)
If a eukaryotic expression vector is to be used as a plasmid, it must also have a eukaryotic origin.
Alternatively, if the vector is designed for integration into the host chromosomal DNA, it must have a sequence for homologous recombination.

6. Introduction of DNA into a Host Cell
Transformation
Introduction of DNA into E. coli or yeast
Transfection
Introduction of DNA into mammalian cells
Transformation of mammalian cells
 becoming cancerous cells
Transformation of yeast (3 techniques)
Removal of cell wall (chemically or enzymatically)  protoplast
Treatment with lithium acetate
Electroporation
Transfection of animal cell
By incubating cells with either calcium phosphate or diethylaminoethyl (DEAE)-dextran
Or by electroporation,
Viruses, lipid-DNA complexes, protein-DNA aggregates are used.

7. Saccharomyces cerevisiae Expression Systems
Advantage of S. cerevisiae
Well known genetics and physiology
Easy to grow
Strong promoters characterized
Naturally occurring 2m plasmid
Posttranslational modification
Secretion of so few proteins
Generally recognized as safe (GRAS) organism
Suitable for production of vaccines, pharmaceuticals

8. S. cerevisiae Vectors Selection markers
Many S. cerevisiae selection schemes rely on mutant host strains that require a particular amino acid (histidine, tryptophan, or leucine) or nucleotide (uracil) for growth.
Use auxotropic strains for the selection marker
Promoters
Inducible promoters
Galactose -regulated promoter: Galactose inducible
(1000-fold increase when induced)
CUP1 (metallothionein) : copper inducible
Constitutive promoters
ADH1 (alcohol dehydrogenase)
GPD (glyceraldehyde-3-phosphate dehydrogenase)

9. Yeast Episomal Plasmid (YEp)
High-copy-number 2μ plasmid
Extensively used for the production of either intra- or extracellular heterologous proteins.
Often unstable

10. Integration of DNA with YIp vector
YIp (Yeast Integrating Plasmid)
The plasmid DNA is linearized because DNA in this form more likely than circular DNA to recombine with chromosome DNA.
Multiple integration into repetitive DNA sequences
d sequence derived from retrotransposon
There are about 400 copies of d sequences.
10 copies of a gene that were inserted into d sequences produced a significant amount of the recombinant protein.

11. Yeast Artificial Chromosome (YAC)
For the cloning of large DNA (> 100kb)
Highly stable and has been used for the physical mapping of human genomic DNA
Not yet been used as an expression system for the commercial products
YAC mimics a chromosome. (Figure 7.6)
ARS: autonomous replicating sequence
CEN: centromere sequence
T: telomere sequence
(tip of chromosome, chromosome stability)
Selectable markers
URA3 ~ uracil synthesis
TRP1 ~ tryptophan synthesis

12. YAC

13. Intracellular Production of Heterologous Proteins in S. cerevisiae
Production of Cu/Zn-SOD
Superoxide dismutase eliminating superoxide radical by generating H2O2 with H2
Administration during blood reperfusion
Therapeutic use for inflammatory diseases
Production of Cu/Zn-SOD in yeast
Proper acetylation of N-terminal Ala

14. Secretion of Heterologous Proteins by S. cerevisiae
Secretory proteins in yeast
All the glycosylated proteins
Containing leader sequence
Addition of leader sequence for secretion
Leader sequence of mating type factor a
Lys-Arg (endoprotease recognition site) adjacent to starting amino acid for proteolytic removal of the leader sequence
Strategies to enhance the secretion of recombinant proteins
Overexpression of PDI (protein disulfide isomerase)
Increase secretion of proteins with disulfide bond

15. Pichia pastoris and Other Yeast Expression Systems
Problems in S. cerevisiae
Plasmid instability
Hyperglycosylation
(more than 100 mannose residues)
Retained within the periplasmic space
(not into the medium)
Ethanol is produced at high cell density,
which is toxic to the cells
Advantages of P. pastoris (Methylotrophic yeast)
Highly efficient and tightly regulated promoter
AOX1 gene promoter, methanol-inducible
No ethanol production, high cell density with the secretion of large quantities of proteins
Simple purification of secreted recombinant proteins
It normally secretes very few proteins.

16. P. pastoris Expression Vector
Most P. pastoris vectors are designed to be integrating plasmids to avoid plasmid instability.
P. pastoris integrating expression vector
3’AOX1
~ a piece of DNA from the 3’ end
of the alcohol oxidase 1 gene
~ for double crossover homologous
recombination

17. Integration of DNA into P. pastoris Chromosome
Single recombination
Dot in the his4 ~ mutation
Double recombination

18. Other Expression Systems
Other yeasts
Hansenula polymorpha (Methylotrophic yeast)
methanol oxidase promoter (MOXp)
Schizosaccharomyces pombe, Candida utili
Aspergillus (filamentous fungus)
commercial production of various enzymes
However, no one system is able to produce an authentic version of every heterologous protein. For this and other reasons, gene expression systems that use insect or mammalian cells have been developed.