Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOC192 > Lecture 20: USING RECOMBINANT DNA TECHNOLOGIES TO MAKE PROTEINS > Flashcards

Lecture 20: USING RECOMBINANT DNA TECHNOLOGIES TO MAKE PROTEINS Flashcards

1
Q

What is the first step of producing a recombinant protein?

A

Isolate gene of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What happens after isolating the gene of interest?

A

Clone into expression plasmin (with correct promoter)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What happens after clones into expression plasmid?

A

Transform into bacteria for expression or isolation of more DNA for use in another expression system

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What happens after transform into bacteria for expression or isolation of more DNA for use in another expression system?

A

Grow cells expressing protein of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What happens after grow cells expressing protein of interest?

A

Isolate and purify the protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is involved in isolating the gene of interest?

A

DNA (with introns)&raquo_space; mRN (with introns)&raquo_space;> cDNA (without introns) using reverse transcriptase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Where is insulin produced?

A

In the pancreas as a pre-proprotein that is further processed by Golgi which won’t happen in bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the solution to insulin needing to be processed?

A

Express chain A and B in separate bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What happens in bacteria 1?

A

Promoter, lac Z gene, insulin gene A subunit, antibiotic resistance gene = insulin A protein accumulates

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What happens in bacteria 2?

A

Promoter, lac Z gene, insulin gene B subunit, antibiotic resistance gene = insulin B protein accumulates

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What happens after the insulin A and B proteins accumulate?

A

Grow the bacteria expressing insulin A and B chains (selected using antibiotic)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the first step of isolating and purifying the protein?

A

Extract and purify lac Z/insulin fusion proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What happens after lacZ/fusion proteins are extracted and purified?

A

Treat with cyanogen bromide to cleave A and B chains (no lacZ)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What happens after the protein is treated with cyanogen bromide?

A

Purify, mix A and B chains to form functional insulin (joined by a disulfide bond)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are the advantages of prokaryotic systems?

A

Relatively low cost, high yield and pathogen free

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the disadvantages of prokaryotic systems?

A

Proteins are often partially folded and the inability to perform post-translational modifications

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are the advantages of recombinant insulin in mammalian cells?

A

Protein can be produced as pre-proprotein and processed efficiently, it will also be secreted from cells for easier purification

18
Q

What is the disadvantage of recombinant insulin in mammalian cells?

A

More expensive to produce

19
Q

What are the differences when making insulin in eukaryotic cells?

A

The promoter is specific to eukaryotic cells, transfected onto eukaryotic cells and there is no need to form disulphide bonds as they are already presens

20
Q

When are some proteins active?

A

Only when post-translationally modified

21
Q

What is an example of glycosylation?

A

Erythropoietin (requires mammalian cells)

22
Q

What does increase in RBC lead to?

A

An increase in the oxygenation of muscle

23
Q

What do muscles use?

A

Oxygen to burn sugar and fats to generate ATP

24
Q

What is required for muscle contraction?

A

ATP

25
Q

What do many disease states result in?

A

lowered RBC count

26
Q

What can chronic renal failure cause?

A

A decrease in EPO levels, leading to anaemia (decreased RBC levels)

27
Q

What can cancer treatments lead to?

A

Anaemia

28
Q

What can administration of recombinant human EPO do?

A

Restore RBC levels

29
Q

When was the EPO gene cloned?

A

In the early 1980’s

30
Q

What is done with the EPO protein?

A

It is post-translationally modified (glycosylation - addition of carbohydrates to asparagine, serine or threonine residues)

31
Q

What is glycosylation of EPO important for?

A

Biological fucntion

32
Q

What is EPO made in?

A

Chinese hamster ovary (CHO) cells

33
Q

What can cells in culture not do?

A

Perform all post-translational modifications equally well, such as gamma carboxylation of glutamate

34
Q

What is gamma carboxylation of glutamate residues a feature of?

A

Many proteins involved in blood clotting

35
Q

What happened in 2006?

A

The first recombinant protein transgenic animal was approved as a drug (anti-thrombin -AT)

36
Q

What may AT deficiency be?

A

Hereditary or acquired

37
Q

What is the frequency of AT deficiency?

A

1 in 2000-5000

38
Q

What does AT deficiency increase the risk of?

A

Inappropriate blood clotting

39
Q

When do AT deficient individuals receive AT?

A

when undergoing surgery or giving birth

40
Q

What is AT protein expressed in?

A

The milk of transgenic goats at lactation then purified from other milk proteins

41
Q

What does the milk specific promoter do?

A

Responds to hormonal signals that induce lactation

BIOC192 (38 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions