K.K Lecture 9&10

Question 1

What are the products of protoplast fusion?

Answer 1

• cybrids
• heterokaryons
• fusants

Question 2

Cybrids

Answer 2

• cytoplasmic fusion
• mitochondrial transfer - restore respiratory competence to petite
• antibiotic resistance - mit. encoded resistance
• unequal amounts of DNA

Question 3

Heterokaryons

Answer 3

• nuclei co-exist in the cytoplasm
• never fuse
• breakdown from fused nuclei

Question 4

Fusants

Answer 4

• true hybrids
• chromosome transfer is the most usual

A (6A) fused with B (6B)
–> (6A + 1B) or (6B + 1A) most unlikely
–> (3A + 3B) or (6A + 6B) unlikely
transfer of chromosomes or genes

Question 5

Biotechnological potential of protoplast fusion

Answer 5

1. increase the rate of production
2. increase the level of some cellular property
3. create novel strains

Legmann and Margalith 1986 fused S.cerevisiae (high fermenter) with S.mellis (sugar tolerance). hybrid combined traits of both parents

Russell et al., 1986 fused S.cerevisiae and S.uvarum. hybrid produced secreted glucoamylase, a less exogenous enzyme required to produce ethanol from starch

Heluane et al., 1993 fused S.cerevisiae and P.tannophilus. hybrid like SC but capable of xylose fermentation

Question 6

Advantages of fungal protoplast fusion

Answer 6

• create novel strains
• hybrids consist of one genome plus one/few chromosomes of the other
• strains have industrial potential
• hybrids may be unstable which leads to a decrease in ploidy
• increase chromosome number which increases productivity

Question 7

Fungal transformation

Answer 7

• yeast transformation: Hinnen, Hicks & Fink, 1978, Saccharomyces cerevisiae protoplasts PEG + Ca^2+
• mechanism of DNA uptake
  1. cell/protoplast fusion
  2. independent of protoplast fusion (plasmid DNA binds to protoplast surface)
• competent cells
  
  1. conversion to protoplasts
  2. lithium acetate treatment
  3. projectile delivery
  4. Agrobacterium mediated delivery

Question 8

Why should we use fungi for recombinant gene expression?

Answer 8

• historical
  - generally regarded as safe
  - widely used
• technological
  - simple to culture
  - fermentation well understood
  - cheap carbon sources
• genetical
  - eukaryotes
  - robust strains
  - small genome
  - natural plasmids (2um)
• molecular biological
  - transformation possible
  - efficient protein secretion
  - splice intons - animal genes
  - RNA polymerase recognise animal promoters

Question 9

Fungi for recombinant protein expression

Answer 9

• advantages
  
  • grow in simple, inexpensive media
  • secret protein into medium
  • capable of post translational modification
• disadvantages
  
  • low level recombinant protein expression
  • altered post-translational modifications

Question 10

Heterologous proteins produced in filamentous fungi

Answer 10

• Aspergillus niger: glucoamylase, proteases, glucose oxidase
• Aspergillus oryzae: lipase, cellulases

Question 11

Heterologous proteins produced in yeasts

Answer 11

• Saccharomyces cerevisiae: glucagon, human papillomavirus vaccine, hepatitis B vaccine
• Pichia pastoris: hepatitis B vaccine, ecallantide

Question 12

Heterologous gene expression in yeast cells

Answer 12

• Gene isolation: DNA from donor.
• Cloning: Put gene into an expression vector.
• Transformation: Vector –> yeast cell.
• Selection: Identify transformants.
• Expression: Transcription & Translation
• Modification: Post-translational chemical modification (glycosylation)
• Signalling: Secretion signal peptides.
• Secretion: Export of protein from cell.

Question 13

Plasmid vectors for transforming yeast cells

Answer 13

• Integrative plasmids:
  
  • Yeast integrative plasmids YIp
• Independently replicating plasmids:
  
  • Yeast replicating plasmid YRp
  • Yeast episomal plasmid YEp
  • Yeast centromeric plasmid YCp
• Specialised plasmid:
  
  • Yeast artificial chromosome YAC
  • Yeast expression plasmid YXp
  • Yeast killer plasmid.

Question 14

Transcriptional promoters in yeast

Answer 14

• Constitutive:
  
  • ADH 1Alcohol dehydrogenase
  • PGK 1 Phosphoglycerate kinase
• Regulated:
  
  • PHO 5 Acid phosphatase
  • ADH 2Alcohol dehydrogenase
  • MFa1/MFa1 Mating pheromones
• Heterologus:
  
  • Viral - SV40
  • Plant - Opaque-2
  • Animal - ARE

Question 15

Selectable genetic markers

Answer 15

• recessive:
  
  • LEU 2, TRP 1, HIS 3, LYS 2: Complement AA auxotrophs
• dominant:
  
  • CUP 1: Copper resistance
    - TUNR: Tunicamycin resistance
    - C220: Chromogenic marker
    or Resistance to drugs - METR

Question 16

Signal sequences

Answer 16

• homologus
  - SUC 2: Periplasmic invertase
  - PHO 5: Periplasmic acid phosphatase
  - Killer toxin: K. lactis
  - Alpha-factor:
• heterologous: Mucor pusillus rennin
  - Chicken lysozyme
  - Bovine prolactin
  - Human gastrin

Question 17

Heterologous protein expression in fungi

Answer 17

• Heterlogous DNA - genomic
  Or mRNA–> cDNA (cDNA can be produced from DNA or mRNA but we need a vector)
• the vectors need to be:
  –> Selectable
  –> Strong promoters (to turn on and ecpress the gene)
  –> DNA sequence to ensure termination of Transcription
  –> DNA sequence to ensure correct Initiation/termination.
• Plasmid in yeast - 2 um Plasmid - naturally occurring.
• Transformation and Selection (to make competent and introduce vector)
  Dominant selection - hygromycin resistance. Auxotrophic strains
  (different selections are used to see what has taken up the plasmid)

Question 18

What are the benefits of S. cerevisiae for heterologous protein expression?

Answer 18

• Rapid growth - easy to culture (easy to grow as it grows at 30 degrees)
• Presence of 2μM plasmid
• Transform intact cells or protoplasts
• Secretes proteins (has the machinery to secret proteins so we can hijack it to secrete our proteins)
• GRAS (generally regarded as safe, so it can be used)
• Expression vectors
  –> YEP based on 2μm - 20-200 copies/cell & 60-95% population (can hijack the proteins and uses as part if the backbone)
  –> High copy number

the problem with this is that: insoluble protein complex in S. cerevisiae (they often end up inside the cell and don’t get expressed or secreted)
to solve this problem we use Protease deficient host for protein expression.

Question 19

Pichia pastoris
(amenable and widely used)

Answer 19

• Employed to produce >100 heterologous proteins
  –> Anti-coagulant (Hirudin variant 2) (to stop clotting in patients with heart attack)
  –> Peptide hormones and vectors
  –> Protein vaccines (hepatitis B vaccine)
• Integrative vectors used (different plasmids can be used)
• Methylotrophic yeast (grows in yeast)
  –> methanol regulates gene expression.

Question 20

Hansenula polymorpha
(unusual, very few others grow at that temp)

Answer 20

• Thermotolerant (43oC) (high temp)
• Multi-copy integration of expression vectors
• Eliminate the need for Methanol (no need for methanol, when the temp is raised gene expression begins, it will screen out other contaminates)

Question 21

Recombinant Hepatitis B virus (HBV) vaccine

Answer 21

• Hepatitis B is a viral infection which attacks liver cells. Causes chronic liver failure, liver cancer and in some cases death.
• Prevented by vaccination
• Originally based on the surface antigen (HBsAg) purified from the blood of chronically infected individuals
• Vaccine produced by growing the live virus in animals and then inactivating it by chemical treatment.
• Led to the risk of infection during manufacture.
• Gene coding for the HBsAg is isolated and cloned into a Vector which is transferred a Yeast Expression system.
• Two most commons yeast species used are Saccharomyces cerevisiae and Pichia pastoris
• The gene is expressed in the yeast and the recombinant protein product of the Hepatitis is obtained.
• The protein is purified and used for vaccination.

Question 22

Use of Fungi for Production of Recombinant Chymosin
- CHEESE MANUFACTURE

Answer 22

• Curdle milk by addition of calcium & Rennet
• Rennet contains proteolytic enzyme chymosin (rennin) (chymosin is produced in young cow stomach only within the first few weeks of life to make cheese. this is a problem as you would have to kill to many cows)
• Isolated from fourth stomach of calves for first few weeks
• Pepsin is produced later.
• Secreted from mucosal cells (ER-GB) as preprochymosin
• Prochymosin: 381-residue precursor molecule MWt: 41,000 Low pH becomes 323-residue enzyme Chymosin (prochymosin allows calf to matabolise milk and protein in milk, it is cleaved to produce chymosin)
• Chymosin (35,600) cleave N-terminal ‘pro’ sequence of 27 A. Acids.
• Chymosin - Aspartyl protease by hydrolysis of k-casein (kappa casein has low abundance protein in milk)
• High milk coagulating activity & low general proteolytic activity.

Question 23

What is the structure of casein micelles?

Answer 23

look at slide 14 lecture 10

Question 24

The problem with Chymosin

Answer 24

• Shortage of Calf Chymosin
  –> Use aspartyl proteases from Mucor miehei or Mucor pussilus but impacts on flavour of older cheeses (it was a problem using chymosin as you would have to slaughter calves at six weeks so they decided to get proteases from fungi)
• Recombinant Chymosin
  –> Commercial Calf Chymosin: purity, quantity & cost (with this there is no longer a need to slaughter calves)

Question 25

What was used initially to solve the chymosin problem?

Answer 25

Initial attempts used E. coli. - Original template mRNA from calf abomasum - Extract RNA, isolate by polyU-sepharose - Large amount of mRNA coded for prochymosin - Prepared cDNA from mRNA after enrichment step. - Insert cDNA into plasmid vector pBR322 - Screen for hybrids ? - Subcloning and sequencing: cDNA carried prochymosin sequence.

Question 26

Cloning in a suitable expression vector

Answer 26

- don't want to produce chymosin in E. coli cytoplasm - harmful - Express prochymosin instead. 1. Fuse prochymosin sequence to N-terminal of LacZ orTrpE prokaryotic sequence initiated transcription & translation 2. Insert prochymosin sequence directly behind prokaryotic promoter (RBS sequence) and ATG codon. - Both approaches lead to prochymosin production = 5% of E. coli protein BUT Prochymosin accumulated in inclusion bodies (it was produced but not secreted and it accumulated in the cells so they used other yeasts to secrete it) - March 1990: FDA approved use of recombinant chymosin Bovine chymosin expressed in E. coli using recombinant DNA Technology.

Question 27

Next step: Produce from Yeasts (Saccharomyces cerevisiae which is a secreting yeast but doesn't do it enough).

Answer 27

- Clone prochymosin gene into YEp-type expression plasmids -- Low yield of soluble protein. - Clone into yeast secretion vectors. Proteins secreted through ER-Golgi pathway similar to animals. - Recombinant plasmid: Strong yeast promoter --> DNA for N terminal AA residues of invertase --> Prochymosin sequence fused to invertase. - Got production of prochymosin but little secretion. Why ? - Some yeasts do not secrete very well (it will produce prochymosin but not secrete it, so they got other secreting yeasts) - Mutate and Select gives higher yield of protein but better approach. - Now use range of good secretors such as: --> Kluyveromyces lactis, Pichia pastoris, Yarrowia lipolytica. - Also used: Trichoderma reesei & Aspergillus awamori.