1) Saccharomyces cerevisiae is a haploid organism that has been intensely studied by genetic analyses for over a century, primarily for the production of beer and bread. This attention has provided a large array of genetic tools and mutant strains for powerful genetic screens.

2) There are a large number of eukaryotic genes that have been evolutionarily conserved between yeast and man. This means that there is likely to be a yeast gene homolog that can be studied by first principle methods.

3) The entire 12 megabase genome of S. cerevisiae has been sequenced, and the corresponding DNA for all ~6,000 protein coding sequences has been mapped, cloned, and mutagenized.

The Internet provides a rich resource of information that can be used as a molecular genetic tool to investigate a wide variety of eukaryotic cell processes. The best place to start is the Stanford University Saccharomyces Genome Database (SGD). One very useful tool that has been developed by the consortium of labs supporting the SGD is the Saccharomyces Genome Deletion Project which has made complete deletions of all ORFs that are 100 amino acids or longer. These deletion strains are available commercially and were made in such a way that unique primer tags are included to facilitate rapid gene identification from a mixture of mutants.

In addition to tools for mining integrated genome databases, you can also find a comprehensive manual on the Genetics and Molecular Biology of Yeast, aka "The Awesome Power of Yeast Genetics" oooh, aaah.

Two basic molecular genetic approaches are used to exploit yeast as a model eukaryotic cell.

1) If the cloned gene has a yeast homolog, it is often possible to investigate gene function using yeast genetics.

2) If the cloned gene has no yeast homolog, but expression of the heterologous gene in yeast results in a reproducible phenotype, then it can be possible to use a "synthetic phenotype" to developed a yeast model.

Representative examples demonstrating the use of yeast models to investigate the function of homologous human disease genes are; 1) the study of the Saccharomyces cerevisiae Met11 gene which is the homolog of Human Methylenetetrahydrofolate reductase (MTHFR) involved in homocysteine metabolism, and 2) analysis of the Schizosaccharomyces pombe genes Yab8p and Yip1p which are homologs to the Human Muscular Atrophy-associated proteins, SMN and SIP1, respectively.

The Flow Diagram below illustrates how you would test the feasibility of using yeast as a model organism to characterize the function of an uncharacterized human ORF containing cDNA.

How similar are the human and yeast genomes?

Lists of ORFs with Mammalian Homologs

Molecular Genetic analyses of apoptosis in yeast is one of the best examples of using Synthetic Phenotypes to investigate protein structure and function. Saccharomyces cerevisea does not appear to undergo classical programmed cell death, nor does the genome contain mammalian homologs for Bcl2/Bax family members or for aspartate-specific cysteine protease (caspases). Nevertheless, regulated expression of the human pro-apoptotic protein Bax in yeast cells results in mitochondrial dysfunction and cell death, suggesting that Bax targets in yeast are similar to those in higher eukaryotes.

Using the Power of Yeast Genetics to identify yeast genes that mediate Bax-induced cell death.

What is the difference between using the "power of yeast genetics" to investigate gene function in a model organism, as compared to using yeast as a molecular genetic tool to identify interacting proteins in a two-hybrid screen? What skills are required for these different approaches to be successful?



Why is the use of yeast genetics to investigate the structure and function of a human gene such a powerful molecular genetic approach, i.e., how is it better than using immortalized cell lines or creating transgenic mice?



If no yeast homolog is identified for a cloned human gene, what experiment is worth trying and how would you interpret the results?



Why might it be prudent to plan your experimental strategy carefully in the event that a null mutation of a mammalian homolog in yeast does not result in a detectable phenotype, what does this result indicate?

Protein expression in cultured cells

High level heterologous protein expression in E. coli is a good source of antigen for antibody production. E. coli expression systems are also used as a ready source of milligram quantities of purified protein for the purposes of crystallographic studies of protein structure. Baculovirus expression vectors to produce eukaryotic proteins that require post-translational modifications to be fully functional.


E. coli expression systems

Most of the E. coli expression vectors used for high level protein production contain regulated promoters that are based on the lac operon. Under optimal conditions, up to 1-10% of the total cellular protein in an IPTG-induced E. coli culture can be the protein product of the cloned gene.

Many types of E. coli expression vectors are designed to produce fusion proteins that contain affinity "tags" at the amino or carboxy terminus of the expressed protein. One example is as shown in figure 2.14. Other

Examples of E. coli expression vectors utilizing fusion proteins with affinity tags are;

1) polyhistidine tag (His)6 that can be used to purify fusion proteins by binding to a Ni2+ affinity column

2) glutathione S-transferase protein moiety that can be used to purify fusion proteins on glutathione affinity column

3) maltose binding protein region that can be purified on amylose resin columns

4) calmodulin binding protein that can be used to purify fusion proteins using calmodulin affinity resin

5) thioredoxin fusion protein that can be purified with thiobond resin.

Each of these fusion proteins encode sequence-specific protease cleavage sites located in the region between the affinity tag and the cloned gene to permit removal of the fusion protein tag following affinity purification.

Fusion proteins made with the E. coli thioredoxin affinity tag at the N-terminus often remain soluble even when expressed at high levels. Expression of the Trx fusion protein is induced by tryptophan-mediated repression of the lambda cI repressor gene integrated into the bacterial chromosome. In tryptophan limiting media, lambda cI repressor levels are high and the PL promoter on the pTrx plasmid is repressed by lambda cI binding to the Po operator site adjacent to PL. Many types of thioredoxin fusion proteins expressed in E. coli localize to the cytoplasmic side of the inner membrane as a consequence of the thioredoxin moiety.

Baculovirus expression systems

The Autographa californica multiple nuclear polyhedron virus (AcMNPV) strain of baculovirus has a genome of 128 kb and normally infects the Lepidopteran alfalfa looper Autographa californica. The AcMNPV host cell line, Sf9, was derived from the ovaries of a Spodoptera frugiperda army worm.

Baculovirus expression systems are used to produce large quantities of recombinant proteins that can be purified using affinity chromatography. In this example, the cloned AMG gene has been inserted into a plasmid shuttle vector that carries the coding sequence for both the baculovirus essential gene ORF 1629, and the 3’ end of lacZ. In vivo recombination in Sf9 cells produces a viable virus that expresses both the AMG gene and the lacZ gene. Infectious virions are harvested from the media and used at a low multiplicity of infection (MOI) in Sf9 cells to isolate individual blue plaques (lacZ+) using agar plates. This baculovirus expression system, called MaxBac, is available commercially from Invitrogen.

Baculovirus vectors are useful for the production of eukaryotic proteins because;

1) baculovirus expressed proteins are often fully active and soluble.

2) the viral genome is large and can accommodate cDNA inserts of up to 15 kb.

3) functional multi-subunit protein complexes can be assembled in vivo and expressed at high levels by co-infecting cells with two or more recombinant viral stocks.

4) the virus has a very restricted host range and is therefore safe to handle and poses minimal environmental risk.

5) under optimal conditions, as much as 1-5 mg of protein can be produced per liter of infected cells.

Why is it important to use a regulated expression system to produce large amounts of recombinant protein in a strain of bacteria (or yeast), why not just use a strong constitutive promoter?



Why isn't it necessary to use regulated expression for high level production of recombinant proteins in the baculovirus system, i.e., how do bacterial and baculovirus systems differ with regard to gene induction?



Why is a recombination strategy required for the development of baculovirus producing SF9 cell line, why not just construct a complete viral vector similar to lambda phage or M13 cloning vectors and use it to infect SF9 cells?



What are the primary advantages and disadvantages of the baculovirus system for protein production?