Chapter 7 Heterologous Protein Production in Eukaryotic Cells

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)

O-linked Glycosylation Yeasts Insects Mammals

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

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.

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.

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

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)

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

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.

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

YAC

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

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

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.

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

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

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.