PROTEIN EXPRESSION IN YEAST

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							             BIOTECHNOLOGY:
              THE EXPRESSION
            OF FOREIGN PROTEINS



COURSE FIGURES CAN BE DOWNLOADED THROUGH
http://www.courseweb.uottawa.ca/BIO4174

I would like to thank Anne Hermans, AAFC, for several slides

                                                          1
BIOTECHNOLOGY: Simple Eucaryotes


 There are many systems for expression. The major ones are

                  •   Escherichia coli *
                  •   Other bacteria
                  •   Yeast *
                  •   Pichia pastoris *
                  •   Baculovirus*
                  •   Animal cell culture
                  •   Plants*
                  •   Animals (sheep, cows, goats)

      * THESE WILL BE THE FOCUS FOR OUR LECTURES       2
BIOTECHNOLOGY: Simple Eucaryotes

 There are many systems for expressing foreign proteins.

 Question: What the general advantages to cloning? (reminder)

  Selection of host and vector

  Can regulate expression by choice of vector

  Genetic modification of host and cloned gene

  Choice of location of product

  Modification of protein produced

  Ease of production and scale

  Can facilitate purification
                                                             3
     Each of these parameters can be modified in each host
BIOTECHNOLOGY: Simple Eucaryotes



   Many of the advantages of E.coli are found yeast
   A single-celled organism, it reproduces asexually and
          sexually(genetics)

   It is easy and cheap to grow on simple food sources. Yeasts
           come in many varieties, grow rapidly and can be
           easily engineered.

    It has a tremendous range of vectors and genetic resources,
           including promoters and regulatory systems.

    We know a lot about yeast, e.g., Saccharomyces cerevisae
         (baking, brewing), Saccharomyces carlsbergensis (brewing)
         and others.

                                                                     4
BIOTECHNOLOGY: Simple Eucaryotes

          Some general “yeast” information
      Single cellular
       fungus
      Eukaryote
      Diploid or haploid
      5 μm diameter

   The classic yeast is
   Saccharomyces
     cerevisiae


                                             5
BIOTECHNOLOGY: Simple Eucaryotes

Saccharomyces cerevisiae as a MODEL SYSTEM
         1997 – first eukaryotic organism sequenced
         6,607 ORF’s (see below)
         Saccharomyces Genome Database
           http://www.yeastgenome.org/

 As of July 13, 2011
                                      Verified ORFs    4932

                                     Uncharacterized
                                                       866
                                         ORFs

                                     Dubious ORFs      809

                                                              6
BIOTECHNOLOGY: Simple Eucaryotes
                    YEAST vs. BACTERIA
  ADVANTAGE YEAST: EUKARYOTIC, thus
      conservation of processes

        Protein folding
        Post translational modifications
        Secretion (proteins targeted to various organelles or
          exported for harvesting (Pichia pastoris does not
          secrete a lot of its own proteins)
        Protein targeting
        DNA replication
        Cell cycle regulation
        Vectors can be maintained as plasmids or integrated.
                                                                 7
BIOTECHNOLOGY: Simple Eucaryotes
                    YEAST vs. BACTERIA

 DISADVANTAGE YEAST:

        More DNA manipulations required (where are these done?)
        Higher number of recombination events
        Longer growth time. Doubling time in bacteria is 20-30 min.
          while in yeast it’s 1.5-2 hours at 30 oC.
        Post-translational modifications such glycosylation may be
          different when compared to human proteins.
          Hyperglycosylation of secreted glycoproteins can be
          observed. In Pichia pastoris, processing can be
          modified to resemble mammalian processing.
                                                               8
BIOTECHNOLOGY: Simple Eucaryotes
              YEAST vs. Other Eucaryotes
       ADVANTAGE YEAST:
       Less expensive, easier to grow, higher throughput
       Shorter Cell Cycle than tissue culture
        ( Human embryonic kidney cells – 1 day doubling)
       Transformation/DNA manipulations easier
       Higher protein yield
       Protein pharmaceuticals free of human disease
       Fewer regulations compared to tissue culture
       More extensive genetics
                                                            9
BIOTECHNOLOGY: Simple Eucaryotes
              YEAST vs. Other Eucaryotes

        DISADVANTAGE YEAST:
        Glycosylation in yeast can be different
        Proteins may get stuck in ER
        It is a lower eukaryote




                                                   10
BIOTECHNOLOGY: Simple Eucaryotes

                    Cloning into Yeast


      When cloning into yeast we need to consider

         Choice of vector

         Choice of transformation system

         Choice of host


                                                    11
BIOTECHNOLOGY: Simple Eucaryotes


         Cloning into Yeast: Choice of Vector

       EPISOMAL (plasmid) – over expression
           High copy (20-100 copies per cell)
           2 μ origin of replication
       INTEGRATIVE – introduce gene into yeast
        chromosome
         Single copy
         understanding protein function/ role in
           pathway
       CENTROMERIC –low copy (YAC)
                                                   12
BIOTECHNOLOGY: Simple Eucaryotes

                   Cloning into Yeast
                Choice of Vector: plasmid
      Bacteria & yeast
       origin of replication
      Antibiotic resistance
       (bacteria)
      Yeast selectable
       marker
          G418,zeocin
           resistance
          Gene for production
           of URA,HIS,TRP,LEU,
           ADE
                                            13
BIOTECHNOLOGY: Simple Eucaryotes

                  Cloning into Yeast
             Choice of Vector: Integrative
           Ability of complementary sequences to
            align and exchange fragments in a double
            crossover event
           Best with linear fragments
           Will work with circular plasmids


    This is a MAJOR advantage of working in yeast
  that has also been used to make deletion libraries.
   This idea is used in many vector/host systems. 14
BIOTECHNOLOGY: Simple Eucaryotes

                 Cloning into Yeast
            Choice of Vector: Integrative
      GCATGCATGCAT                 GGCCAATTGGCC   yeast
      CGTAGCTACGTA                 CCGGTTAACCGG

      GCATGCATGCAT                 GGCCAATTGGCC
      CGTAGCTACGTA         YFG     CCGGTTAACCGG




     GCATGCATGCAT                  GGCCAATTGGCC
     CGTAGCTACGTA                  CCGGTTAACCGG


    GCATGCATGCAT                   GGCCAATTGGCC
    CGTAGCTACGTA          YFG      CCGGTTAACCGG   yeast
                                                      15
BIOTECHNOLOGY: Simple Eucaryotes

                    Cloning into Yeast
                     Choice of Host
       Defects in genes used for selectable markers
       Protease deficient strains
       Yeast ORF collection
       Knock-out mutants of functional homologues
       TAP (Tandem Affinity Purification) tagged
        collection
            For more on TAP, see http://www.embl-
        heidelberg.de/ExternalInfo/seraphin/TAP.html


                                                       16
BIOTECHNOLOGY: Simple Eucaryotes

    Cloning into Yeast: constitutive promoters

       Constitutive promoters

           ADH1 – alcohol dehydrogenase
           GPD – glyceraldehyde-3-phosphate
            dehydrogenase
           PMA1 – plasma membrane H+-ATPase
           PDR5 – pleiotropic drug resistant pump

       PMA1 & PDR5 – expression can reach up to
        10% of plasma membrane proteins


                                                     17
BIOTECHNOLOGY: Simple Eucaryotes

       Cloning into Yeast: inducible promoters

      Inducible promoters:

          Gal1, the major one (see next page)
          CUP1 – metalothionein gene promoter
             Inducible by addition of copper ions

          PHO 5 – induced by low extracellular inorganic
            phosphate
          HSE – tandem heat shock elements
             Induced by increase of temperature to 37 C
                                                       o




                                                            18
BIOTECHNOLOGY: Simple Eucaryotes

      Cloning into Yeast: inducible promoters
        Gal1
           GAL genes – metabolism of galactose

           Inducible by galactose

           Repressed in presence of glucose




                                                  19
BIOTECHNOLOGY: Simple Eucaryotes
             Cloning into Yeast: VLP System
   An alternative to these systems using the yeast VLP
   (virus-like particle) has been developed by Kingsman.
   A VLP if the yeast equivalent of a retroviral intermediate.
   Kingsman AJ, et al. (1995) Yeast retrotransposon particles as antigen delivery
      systems. Annals of the New York Academy of Sciences 754:202-13.




                                     Yeast with purified HIV p24-VLPs
                                                                                    20
                                              (remember M13?)
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast (example)
Functional expression of the Human mdr1 gene
        Mdr1 = p-glycoprotein
        170 kD plasma membrane glycoprotein
        ATP-driven drug exporting pump
        Homologues in bacteria, yeast(prd5), humans, plants
        First cloned by a Canadian, Dr. V. Ling (Nature 316: 817-819)




                                                                         21
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast (example)
Functional expression of the Human mdr1 gene

        Mdr1 gene product was localized to the plasma
         membrane
        Mdr1 binds ATP
        Mdr1 NOT glycosylated
        Functional Assay

        Expression can be improved by using a chaperone
         ( Figler et al. 2000 Archives of Biochem. &
         Biophysics 376:34-46), protease deficient strain,
         different promoter
                                                             22
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast (example)
Functional expression of the Human mdr1 gene


125 uM valinomycin




 150 uM valinomycin
Equal number of cells
                                           23
 BIOTECHNOLOGY: Simple Eucaryotes

               Cloning into Yeast
 Functional expression of the Human mdr1 gene

Control




Clone




          Phase contrast            Rhodamine 6G flourescence   24
BIOTECHNOLOGY: Simple Eucaryotes

          Cloning into Yeast: Pichia pastoris
       Like Saccharomyces cerevisiae:
          Easy to manipulate

          Faster, easier, less expensive than other
           eukaryotic systems
       Advantage over Saccharmoyces cerevisiae:
          10-100 fold higher heterologous protein
           expression levels!!
Pichia is a methylotrophic yeast(can metabolize MeOH)
                         AOX
            CH3OH                     HCHO

                       O2      H2O2                    25
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast: Pichia pastoris
        2 genes coding alcohol oxidase:
           AOX1 and AOX2




        AOX1 responsible for majority of activity
        AOX1 inducible by methanol
        Can reach up to 30% total soluble protein in cells
         grown with methanol
        Controlled at transcription level
        Can make up 5% polyA RNA
        Similar to Gal1 promoter:

     Glucose represses transcription, even in presence of
                           methanol                           26
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast: Pichia pastoris
        Pichia Expression Vector




                                               27
BIOTECHNOLOGY: Simple Eucaryotes

            Cloning into Yeast: Pichia pastoris

    Advantages for P. pastoris
       Grows to extremely high cell densities
       Intracellular or Secreted
           Pichia secretes low levels of native protein
           Easier purification!
       High product yields
           Example: Sea Raven anti-freeze protein
              40 mg/L (secreted)

           Example: Hepatitis B virus surface antigen
              Yield = 400 mg/L (intracellular)

                                                           28
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast: Pichia pastoris
   The problem of inappropriate gycosylation




       This problem is not restricted to Pichia,
                                                   29
               neither are the solutions
BIOTECHNOLOGY: Simple Eucaryotes

         Cloning into Yeast: Pichia pastoris
          Solution 1: there is no problem

        Insulin – therapeutic protein expressed in
         E.coli and yeast
           No glycosylation required




        Majority of human proteins do require
         glycosylation (but may have other
         modifications)

                                                      30
BIOTECHNOLOGY: Simple Eucaryotes

          Cloning into Yeast: Pichia pastoris
            Solution 2: engineer the host.

       Knockout endogenous glycosylation reaction
            (och1: alpha 1-6 mannosyltransferase)
       Introduce active human pathway
            mannosidases I and II
            N-acetylglucosaminyl transferases I and II

            uridine 5'-diphosphate (UDP)-N-acetylglucosamine

                    transporter.
       Verify by introducing human reporter gene K3
                    (has single glycosylation site)
                                                                31
BIOTECHNOLOGY: Simple Eucaryotes

           Cloning into Yeast: Pichia pastoris
             Solution 2: engineer the host.
       Isolated several yeast strains showing high yield of reporter human
          K3 protein with human-like glycosylation




Hamilton, SR (2003) Production of Complex Human Glycoproteins in Yeast.
Science 301: 1244 - 1246. This paper describes the “humanization” of
the Pichia glycosylation pathway.                                            32
BIOTECHNOLOGY: Simple Eucaryotes
           Cloning into Yeast: products

    Product               Protein              Host
    Novolin PenFill       Insulin              Saccharomyces
    Cartridges                                 cerevisiae
    Hepatitus B Vaccine   Hep B virus     Pichia pastoris
                          surface antigen

    GAVAC vaccine         Bm86 tick            Pichia pastoris
    against cattle tick   protein

    Leukine               Granulocyte-         Saccharomyces
                          macrophage
                          colony stimulating   cerevisiae
                          factor

                                                                 33
BIOTECHNOLOGY: Other important Eucaryotes



        Other Yeast Expression Systems

           Hansenula polymorpha
           Kluyveromyces lactis
           Schizosaccharomyces pombe
           Schwanniomyces occidentalis
           Yarrowia lipolytica

           Trichoderma reesei (Iogen, Ottawa)
                                                 34
BIOTECHNOLOGY: Simple Eucaryotes
            Industrial Enzymes Produced:
          Xylanase – breaks down hemicellulose
              pulp and paper,
              textile industry
              animal feed
          Cellulase – breaks down lignin/celluose
              animal feed
              ethanol production
          Genes used: native to Trichoderma
          Modified by Directed Evolution
          Re-introduced into the fungus
          Yield: 60 g/L !!!!!!!!!!!!!               35
BIOTECHNOLOGY: Simple Eucaryotes



        SUMMARY
           As a unicellular eukaryote, yeast is quick, easy and
            inexpensive to genetically manipulate and culture
           Yeast share many conserved pathways with higher
            eukaryotes making it an excellent platform for
            studying protein function
           As well, the wealth of knowledge and set of tools
            available for Saccharomyces cerevisiae, make it a
            very powerful genetic tool for studying protein
            function
           High protein yield and ease of industrial scale-up,
            make other yeast/fungal strains useful for
            pharmaceutical protein production

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BIOTECHNOLOGY: Other important Eucaryotes




                BIOTECHNOLOGY:
                 THE EXPRESSION
               OF FOREIGN PROTEINS
                        in
                   BACULOVIRUS


   COURSE FIGURES CAN BE DOWNLOADED THROUGH
   http://www.courseweb.uottawa.ca/BIO4174
                                            37
BIOTECHNOLOGY: Other important Eucaryotes
        BIOTECHNOLOGY: Expression in Baculovirus


 Course Map            Advantages of insect cells

 Plasmid Vectors        High level of expression in cells or animal
 Viral Vectors
 Specialized Vectors        (up to 30%).
 Escherichia coli *
 Other Bacteria         Correct folding.
 Yeast *
 Pichia pastoris *
 Baculovirus            Proteins can be targeted to organelles or
 Cell Culture               exported outside for harvesting
 Plants*
 Animals
                        Post-translational modifications “similar”
                             to those in mammalian cells and
                            being “improved” by engineering.
                                                                 38
BIOTECHNOLOGY: Other important Eucaryotes

       BIOTECHNOLOGY: Expression in Baculovirus
    Disadvantages

     More difficult to work with. Specialists.

     Slow generation time

     Not suitable for proteins with repetitive sequences

     Culturing is costly (compared to bacteria, yeast)
         but less than for mammalian cells, e.g, used for
         potential AIDS vaccine (HIV glycoproteins)

     Must confirm all processing steps, since differences
         may be protein-specific.
                                                             39
BIOTECHNOLOGY: Expression in Baculovirus




BEVS(Baculovirus Expression Vector System)provides a good balance between different
decision categories. http://www.proteinsciences.com/technology/technology_why.htm     40
BIOTECHNOLOGY: Other important Eucaryotes
     BIOTECHNOLOGY: Expression in Baculovirus

Properties of Baculovirus:

Rod shaped viruses that infect mostly insects
      40-50 nm in width and 200-400nm in length.

Genome is a large, ds, circular DNA of 80-200kb.

Replicate in nucleus

Common ones are

Autographa californica (AcNPV) NC_001623 133,894 bp
Bombyx mori nucleopolyhedrovirus (BmNPV) L33180 128,413 bp


                                                              41
BIOTECHNOLOGY: Other important Eucaryotes
       BIOTECHNOLOGY: Expression in Baculovirus
   Common hosts for Baculoviruses



                    Spodoptera frugiperda
                    (fall armyworm)




      Trichoplusia ni ( cabbage looper )    Bombyx mori (common silkworm)




                                                                      42
BIOTECHNOLOGY: Expression in Baculovirus

                      The life cycle of baculovirus.




                                                       43
http://www.mygene.net/Research/images/baculo.jpg
BIOTECHNOLOGY: Other important Eucaryotes
BIOTECHNOLOGY: Expression in Baculovirus

Important for Genetic Engineering

Can form inclusion bodies or polyhedra within the infected
     cell for long term storage. An inclusion body is a mass
     of carbohydrate that surrounds the nucleoplasmid and
     50% of the mass is the polyhedron protein.

       a very strong promoter (polh) for expression
       since late, have the potential for accumulating
              toxic proteins

Do not see polyhedra in culture

       ease of purification
                                                               44
BIOTECHNOLOGY: Expression in Baculovirus




  Slide from A. Weber, Cambridge, UK.      45
BIOTECHNOLOGY: Other important Eucaryotes

   BIOTECHNOLOGY: Expression in Baculovirus
Important for Genetic Engineering

In some cases high expression can overwhelm the
      host’s protein processing system.

       switch to a regulated or less powerful early promoter

The genome is very large

       can accommodate very large genes and proteins
       makes it difficult to clone by regular restriction/ligation.

            HOW HAVE COMPANIES OVERCOME
                  THESE PROBLEMS?                               46
BIOTECHNOLOGY: Other important Eucaryotes




          COMPANIES CLONE INTO SMALL,
          TRANSFER VECTORS AND USE
          HOMOLOGOUS RECOMBINATION!!

       Homologous recombination between BAC DNA and a

transfer vector containing YOUR gene creates a viable virus

with YOUR gene that is genetically homogenous
            (does this remind you of yeast?)


                                                              47
BIOTECHNOLOGY: Other important Eucaryotes
BIOTECHNOLOGY: Expression in Baculovirus


         Baculovirus (AcMNPV) Cloning Process
          Transfer vector


          Cloned gene
    5’                       3’
     x                       x                Cloned gene
                                      5’                    3’


           Polyhedrin gene

          AcMNPV DNA                       Recombinant
                                           AcMNPV DNA

           This should remind you of yeast!                      48
BIOTECHNOLOGY: Other important Eucaryotes

      Proteins expressed in Baculoviruses include
 Protein                                Use
 α and β interferon                     Cytokines; antivirals
 Adenosine deaminase                    SCIDs
 Erythropoietin(EPO)                    RBC production.
 Interleukin 2                          Immune modulation
 Poliovirus proteins                    Vaccine
 Tissue plasminogen activator(TPA)      Dissolves clots

 “To date, over a thousand proteins have been expressed using the BEVS,
 with 98% being biologically active.”

 Recent Proteins (human) expressed in this system:
 Renin, hepatocyte growth factor, tryptase, squalene synthase, interleukin-5,
 α-1,3-fucosyltransferase, proapoA-I, nerve growth factor, tumour necrosis
 factor-β, erythrocyte anion exchanger, β-glucuronidase, leukotriene A4
 hydrolase, plasminogen, leptin, thyroid peroxidase etc.
                                                                          49
BIOTECHNOLOGY: Other important Eucaryotes

 Of particular interest is the ability to rapidly clone antigens
 such as

 SARS:
 Mortola,E. & Roy, P. (2004) Efficient assembly and release of SARS
 coronavirus-like particles by a heterologous expression system.
 FEBS Letters 576(1-2): 174-178.

 and Influenza
 Brett, IC. & Johansson, BE. (2005) Immunization against influenza A
 virus: comparison of conventional inactivated, live-attenuated and
 recombinant baculovirus produced purified hemagglutinin and
 neuraminidase vaccines in a murine model system. Virology 339(2):273-80.

 Nwe, N.et al.(2006) Expression of hemagglutinin protein from the avian
 influenza virus H5N1 in a baculovirus/insect cell system significantly
 enhanced by suspension culture. BMC Microbiology. vol. 6.
                                                                          50
BIOTECHNOLOGY: Other important Eucaryotes

  Data from Nwe, N.et al.(2006)




 Nuclear localization of rHA1   Expression of rHA1 in baculovirus infected
 from avian influenza H5         cells. Monolayer (A) and
                                suspension (B) culture conditions.
                                                                        51
BIOTECHNOLOGY: Other important Eucaryotes

 Future Developments for Baculoviral Systems



“Humanize” the protein
processing system

Grow baculovirus vectors
on cells for expression
and proper processing




                                               52
BIOTECHNOLOGY: Other important Eucaryotes




                                                                       Choice of
                                                                       insect or cell
                                                                       culture systems




                                                                          “Human”
                                                                          modifications


                                                                          Equivalent to
                                                                          M13 phage
                                                                          display!!
Figure 1 of Kost et al. (2005) Baculovirus as versatile vectors for protein         53
expression in insect and mammalian cells. Nature Biotechnology 23: 567 - 575.
BIOTECHNOLOGY: Other important Eucaryotes



       Relationship of the section “Other Important
                        Eucaryotes”
               to your the NSERC projects
       Can lead to higher accumulation of your protein

       Intermediates for rapid screening

       Intermediates for assembly of genes/pathways
         prior to transformation of plant/animal.




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