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Bio chem > Lecture 20 - Recombinant proteins > Flashcards

Lecture 20 - Recombinant proteins Flashcards

1
Q

Key 5 steps in producing a recombinant protein

A
  1. Isolate gene of interest
  2. Clone into expression plasmid –
  3. Tranform into bacteria for expression or isolation of more DNA for use in another
    expression system
  4. Grow cells expressing protein of interest
  5. Isolate and purify the protein
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2
Q

Advantages of prokaryotic systems

A

relatively low cost
high yield
pathogen free

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3
Q

Disadvantages of prokaryotic systems

A

proteins often partially folded

inability to perform post-translational modifications

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4
Q

Recombinant Insulin production Step 1

A

Reverse transcriptase

cDNA = no introns

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5
Q

Insulin is produced in

A

pancreas as pre-proprotein further processed by Golgi
– won’t happen in bacteria

Solution = express
chain A and B
separately in bacteria

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6
Q 
Insulin Production
Step 1
Step 2/3
Step 4
Step 5
A

Recombinant Insulin production.
Clone gene into expression plasmid(s) and transform bacteria.
Grow bacteria expressing insulin A and B chains.
Extract and purify.

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7
Q

Step 2-3 Clone gene into expression plasmid(s) and transform bacteria

A

lac Z gene
Promoter
Antibiotic resistance gene
Insulin Gene A or B subunit

Transforms in E. Coli host

lac Z / Insulin A or B fusion protein accumulates in cell.

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8
Q

Protein purification

A

Recombinant insulin

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9
Q

Step 5. Extract and purify

A

lac Z / insulin fusion proteins

Treat with Cyanogen bromide to cleave A and B chains

Purify mix A and B chains to form functional insulin

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10
Q

Why Make recombinant insulin in mammalian cells?

A

– Protein can be produced as
a pre-pro-protein and processed efficiently

– Will be secreted from cells (easier purification)

– More expensive to produce.

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11
Q

Making recombinant insulin in eukaryotic cells 5 Steps

A
  • Step 1. Isolate cDNA for insulin
  • Step 2. Clone into eukaryotic expression plasmid
  • Step 3. Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
  • Step 4. Extract recombinant insulin from cell media
  • Step 5. Purify insulin
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12
Q

Making recombinant insulin in eukaryotic cells

Step 1.

A

Isolate cDNA for insulin

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13
Q

Making recombinant insulin in eukaryotic cells

Step 2

A

Clone into eukaryotic expression plasmid

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14
Q

Making recombinant insulin in eukaryotic cells

Step 3

A

Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells

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15
Q

Making recombinant insulin in eukaryotic cells

Step 4.

A

Extract recombinant insulin from cell media

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16
Q

Making recombinant insulin in eukaryotic cells

Step 5.

A

Purify insulin

17
Q

Some proteins are only active when

A

post-translationally modified

18
Q

recombinant human proteins

A

Therapeutic proteins –

19
Q

Glycosylation

A

requires mammalian cells e.g. erythropoietin = EPO

20
Q

EPO as a performance enhancing drug – blood doping effects

A

increase RBCs
increase in oxygenation of muscle

muscle uses oxygen to burn sugar and fats to generate
ATP

ATP required for muscle contraction

21
Q

Why would we want to make recombinant EPO?

A

restore RBC levels

lowered RBC counts.

chronic renal failure decrease in EPO levels, leading to anaemia (decrease
in RBC levels)

cancer treatments (chemotherapy) may lead to anaemia

22
Q

Erythropoietin (EPO) gene cloned in 1980s

A

protein is post-translationally modified (glycosylation)

made in Chinese Hamster Ovary (CHO) cells

23
Q

glycosylation

A

Addition of carbohydrates to asparagine, serine or threonine residues.

important for biological function.

24
Q

Pharming:

A

using whole animals to make recombinant proteins

25
Q

Expression vector for EPO

A

Promoter – for transcription in mammalian cells

Human EPO

Protein purification

26
Q

cells in culture cannot perform all

A

post-translational modifications equally well

e.g. γ carboxylation of glutamate

27
Q

what is a feature of many

proteins involved in blood clotting?

A

γ-carboxylation of certain glutamate residues

28
Q

anti-thrombin (AT)

A

2006 (Europe) 1st recombinant protein transgenic animal was approved as a drug

29
Q

AT deficiency

A

hereditary or acquired

1 / 2000-5000

increase risk of blood clotting

30
Q

deficient individuals receive AT when

A

undergoing surgery or giving birth

31
Q

AT protein expressed in

A

milk of transgenic goats at lactation

AT then purified from other milk proteins

32
Q

milk specific promoter

A

responds to hormonal signals that induce lactation

33
Q

Recombinant DNA technologies are important for

A

production therapeutic proteins

provide a safe means of getting human proteins for clinical use

34
Q

Eukaryotic systems must be used

A

for proteins that require post-translational modification for their activity

35
Q

There are a variety of cell systems available for

A

producing recombinant proteins

Bio chem (36 decks)

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