Lecture 3 Flashcards

1
Q

What does gram negative bacteria mean?

A

Gram negative bacterias does not get stained during the gram staining method, while gram positive bacterias does. The difference between positive and negative is the difference cell walls, where gram positive have a thick layer of peptidoglycan that gets stained.

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2
Q

What different kind of sites does a typical plasmid vector have?

A
  1. Origin
  2. Selection marker
  3. Promoter
  4. Shine-Dalgarno (Ribosome binding site)
  5. N-terminal tag/fusion protein
  6. Multi-cloning site
  7. Restriction site
  8. Gene insert
  9. Terminator
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3
Q

What kind of information does a general vector contain regarding the specific protein that will be produced in the cell?

A
It contains the information for:
1. Where
2. When
3. How
the specific protein will be produced in the cell
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4
Q

Can E. coli read genes of any organism?

A

???

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5
Q

What are introns?

A

Introns are the non-coding parts of a gene that is transcribed but then is removed during RNA splicing, before translation.

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6
Q

What three problems might prevent efficient expression of a foreign gene cloned in E.coli?

A
  1. E. coli can’t excise introns (a problem since E. coli don’t contain introns so the bacteria doesn’t have the necessary machinery for removing introns from transcripts)
  2. Premature termination of transcription (the sequences might be unproblematic in normal host cells but can act as a terminator in bacteria)
  3. Codon bias (almost every organism use the same genetic code but each organism has a bias toward preferred codons. This bias reflects the efficiency with which the tRNA molecules in the organism are able to recognize the different codons. If a cloned gene contain a lot of disfavored codons, the E. Coli might tRNA may encounter problems which means the amount of synthesized protein is reduced.) the result of this is that E. coli has difficulty translating the proline codons in a human gene.
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7
Q

What problem can be caused by secondary structure at the start of an mRNA?

A

The structure can interfere with the ribosome binding site by covering it.

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8
Q

How can you synthesize genes?

A

Algorithms can be used to optimize the sequence for expression in a given host and can then be synthesized chemically.

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9
Q

What are codon optimizations normally based on?

A
  1. tRNA abundance in the specific host
  2. Codon bias
  3. Avoid secondary mRNA structures
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10
Q

What is the level of expression dependent on?

A

The strength of the promoter.

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11
Q

What does a strong respectively weak promoter mean in terms of number of proteins translated?

A

A strong promoter leads to many transcripts which leads to many protein molecules translated.
A weak promoter leads to few transcripts which leads to a low number of protein molecules.

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12
Q

Can you regulate the promoter? What are these types of gene regulations called?

A
  1. An inducible gene

2. A repressible gene

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13
Q

How does a inducible gene work? An example

A

An inducible gene is normally of but when a regulatory chemical binds/enters, the gene switches on.

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14
Q

What is a repressible gene?

A

A repressible gene is a gene that is normally on but when a regulatory chemical enters/bind, the gene switches off.

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15
Q

Describe how the lac promoter works. Is it a inducible gene or repressible gene?

A

The lac promoter is the sequence that controls the transcription of the lacZ gene coding for beta-galactosidase. The lac promoter is induced by IPTG so when this chemical is present it switches on the transcription of the gene.
When lactose is present it binds to the repressor and the repressor let’s go of the operator and let the RNA-polymerase bind to the promoter and start the transcription.

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16
Q

What are hybrid protein generated for?

A

They are generated to facilitate gene expression and/or add new functionality to the protein.

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17
Q

How is a hybrid gene constructed and how is it synthesized?

A

The foreign gene is inserted after the start of an E. coli gene, which creates a hybrid gene that starts with E. coli and ends with the foreign gene, without a break into codons between the two different genes. The product of gene expression is therefore a hybrid or fusion protein, consisting of the short peptide coded by the E. coli reading frame fused to the amino-terminus of the foreign protein.

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18
Q

What four advantages does the fusion system have?

A
  1. Efficient translation of the mRNA produced from the cloned gene depends not only on the presence of a ribosome-binding site but is also affected by the nucleotide sequence at the start of the coding region. This is probably because the secondary structures resulting from intrastrand base pairs could interfere with attachment of the ribosome to its binding site. This possibility is avoided if the pertinent region is made up entirely of natural E. coli sequences.
  2. The presence of the bacterial peptide at the start of the fusion protein may stabilize the molecule and prevent it from being degraded by the host cell. In contrast, foreign proteins that lack a bacterial segment are often destroyed by the host.
  3. The bacterial segment may constitute a signal peptide, responsible for directing the E. coli protein correct position in the cell. If the signal peptide is derived from a protein that is exported by the cell, the recombinant protein may itself be exported, either into the culture medium or into the periplasmic space between the inner and outer cell membranes. Export is desirable because because it simplifies the problem of purification of the recombinant protein from the culture.
  4. The bacterial segments may also aid purification by enabling the fusion protein to be recovered by affinity chromatography. For example, fusions involving the E. coli glutathione-S-transferase protein can be purified by adsorption onto agarose beads carrying bound glutathione.
    According to slide 14:
  5. Stabilize mRNA molecules —> prevent degradation
  6. May have a signal peptide for correct targeting in the cell (secretion)
  7. Easier purification
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19
Q

What can leader peptides be used for?

A

To direct where the protein ends up.

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20
Q

What is inclusion bodies (IB)? Can it be a problem? How are they formed?

A

IB is a crystalline or paracrystalline deposit within a cell, often containing substantial quantities of insoluble protein. IB can be a major problem for production of recombinant proteins. IBs are formed by misfolded or unfolded proteins, which aggregates into non-functional protein clusters that are excluded by the cell’s quality control system.

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21
Q

What is protein folding often aided by?

A

Chaperones. (Hsp70 proteins in eukaryotes).

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22
Q

What is a non-integrative plasmid?

A

Plasmids that are able to multiply within the cell independently of the main bacterial chromosome.

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23
Q

What is an episome? What is an advantage of this?

A

An episome, or integrative plasmids, insert themselves into the bacterial chromosome to be able to replicate. Chromosomal integration are often more stable.

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24
Q

What are some reasons that long term continuous production mode may face some losses? What do we actually want?

A
  1. Loss of plasmids
  2. Down-regulated transcription/translation

We want a high concentration during the generation time. What we might get during “normal” production is concentration loss during the generation time.

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25
Q

Is heterologous protein production a significant burden to the cell?

A

Yes.

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26
Q

What five steps are there for creating a production strain?

A
  1. Build the vector
  2. Transformation
  3. Identify colonies that produce protein
  4. Analyze under process conditions
    - purity and quality
    - yield (g/g)
    - productivity (g/L/h)
    - titers (g/L)
    - stability of production strain
  5. Learn and re-design
  6. See step 1.
27
Q

What six different kind of expression hosts can you choose from?

A
  1. E. coli
  2. Yeast
  3. Mammalian cells
  4. Insect cells
  5. Plant cells
  6. Transgenic animals
28
Q

Does a universal expression host exist?

A

No.

29
Q

What does optimal protein production system depend on?

A
  1. Properties of the specific protein (Can it be functionally expressed in the host? Does it require difficult PTMs?)
  2. Required quality (What will the recombinant protein be used for? How pure does it need to be?)
  3. Quantity (How much of the protein is required? What is the required scale for the production process?)
  4. Price (What is the selling price of the protein? How much is the downstream process allowed to cost?)
    etc
30
Q

What is PTMs?

A

Post-translational modifications. The covalent and enzymatic modification of proteins after translation.

31
Q

What kind of PTMs are there?

A

Many. Like glycosylation, S-S bridges, proteolytic cleavage, phosphorylation etc

32
Q

What kan incorrect PTMs lead to in biopharmaceuticals?

A

Activation of immune system.

33
Q

What determine which system that can be used for recombinant protein production?

A

PTM requirements.

34
Q

What is downstream processing?

A

Downstream processing is the recovery and purification of biosynthetic products from natural sources, like animal or plant tissue or fermentation broth etc.

35
Q

Downstream processing is expensive. What is important in broth solution?

A

Titers.

36
Q

What group of microorganisms can assimilate methanol as carbon source for growth?

A

Methylotropic.

37
Q

Why is it a problem that S.cerevisiae can carry glycans?

A

It’s because yeast glycans are hyperglycosylated - they contain many more sugar units than a mammalian glycan. This is a problem because a hyperglycosylated protein is more likely to be recognized as foreign if injected into an animal and will therefore cause an immunogenic reaction.

38
Q

What is shuttle vectors?

A

Shuttle vectors are vectors constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli, then used in a system which is more difficult or slower to use, like yeast.

A cloning device/vector that functions in more than one system. You can initaite your cloning process in E.coli and after you’ve finetuned the DNA in E.coli, you can take the DNA without modifiying it further and simply isolate the shuttle vector and then put it into a different organism.

39
Q

Where can shuttle vectors replicate?

A

Shuttle vectors can replicate in both prokaryotes and eukaryotes.

40
Q

Which parts are needed in a yeast shuttle vector?

A

See slide 28.

41
Q

What three main types of yeast plasmids are there? Describe them shortly.

A

See page 124.

  1. YEp: Yeast episomal plasmid (These are most similiar to bacterial plasmids and considered high copy. A fragment from the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propogate per cell. This plasmid can be replicated as an independent plasmid, but it can also be integrated into one of the yeast chromosomes. Integration occurs because the gene carried on the vector as a selectable marker, is very similar to the mutant version of the gene present in the yeast chromosomal DNA).
  2. YRp: Yeast replicative plasmid (These vectors contain an ARS origin (Autonomously Replicating Sequence) derived from the yeast chromosome. These vectors can replicate independently of the yeast chromosome, though they often tend to be unstable and may be lost during budding. 5-100 copies/cell).
  3. YIp: Yeast integrative plasmid (These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination. 1 copy/cell).
42
Q

What is auxotrophic markers?

A

Auxotrophc marker is defined as a wild-type allele of a gene that encodes a key enzyme for the production of an essential monomer used in biosynthesis. See page 123

43
Q

Compare the four yeast plasmids in regards of ORI, Transf eff., number of copies, status in the cell and stability.

A

See slide 34.

44
Q

What are immortilised cells?

A

Cells that can undergo “unrestricted” number of divisions.

45
Q

Can mammalian cells undergo PTMs without problem?

A

Yes.

46
Q

Can mammalian cells do proper folding?

A

Yes.

47
Q

Can mammalian cells do efficient cleavage of signal peptides?

A

Yes.

48
Q

What are four cell lines that are used for recombinant protein production?

A
  1. Chinese hamster overy (CHO) cells
  2. Baby hamster kidney (BHK) cells
  3. NS0 murine myeloma cells
  4. Human embryonic kidney (HEK) cells
49
Q

What is the genome called that is used by viruses as cloning vectors?

A

The SV40 genome.

Lite oklart vad detta innebär dock.

50
Q

What is pharming?

A

Pharming is where a transgenic organism acts as the host for protein synthesis.

51
Q

What is a trangenic animal?

A

It’s an animal that contains a cloned gene in all of its cells.

52
Q

How can a transgenic mouse be produced? Can this be used for other animals?

A

A transgenic mouse can be produced by microinjection of the gene to be cloned into a fertilized egg cell. This works well with mice but the injection of fertilized cells is inefficient or impossible with many other mammals.

53
Q

What method can be used for other animals than for mice? How does it work?

A

Generations of transgenic animals for recombinant protein production is usually called nuclear transfer. The nucleus is removed from an oocyte and the nucleus of a transgenic somatic cells is inserted in the oocyte.
After insertion into a foster mother, the engineered cell retains the ability of the original oocyte to divide and differentiate into an animal, one that will contain the transgene in every cell.
This is a lengthy procedure and transgenic animals are therefore expensive to produce, but the technique is cost-effective because once a transgenic animal has been made it can reproduce and pass its cloned gene to its offspring.

54
Q

Why are farm animals often a successful approach when producing “transgenic proteins”?

A

Because farm animals produce a lot of milk and the since the cloned gene is attached to the promoter for the animals beta-lactoglobulin gene, the recombinant protein will be secreted in the milk.

55
Q

Why is the production of human proteins often correctly modified in pigs and sheep?

A

Because they’re mammals.

56
Q

What are the advantages of using bacteria as a production host? And the disadvantages?

A

+ Easy to genetically engineer
+ Lowest cost
+ Highest yield
- Least PTM

57
Q

What are the advantages of using yeasts as a production host? And the disadvantages?

A
\+ Easy to genetically engineer
\+ Low cost
\+ High yield
\+ Good PTM for certain proteins
- Longer time
- Less PTM
58
Q

What are the advantages of using insect cells as a production host? And the disadvantages?

A

+ Relatively higher yield
+ Good PTM
- Longer time
- Lower yield

59
Q

What are the advantages of using animal cells as a production host? And the disadvantages?

A
\+ Natural protein configuration
\+ Best PTM
- Longer time
- Highest cost
- Lowest yield
60
Q

Why do insect cells provide an alternative to mammalian cells for protein production?

A

Because insect cells does not behave any different in cultures compared to mammalian cells, and they have the advantage of, thanks to a natural expression system, provide high yields of recombinant protein.

61
Q

What is the expression system in insect cells based on? What kind of gene does this viruses genome include?

A

Baculoviruses. It includes the polyhedrin gene, whose product accumulate in the insect cell in large nuclear inclusion bodies.

62
Q

How much of the total cell protein does polyhedrin make up?

A

Up to 50 %.

63
Q

Why are insect cells useful when using recombinant protein?

A

Because the polyhedrin gene can be replaced by one for a recombinant protein, and it will thus produce similar level s of the desired protein.