Brainscape's Knowledge GenomeTM

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


See full index

Techniques in biomolecular science > Lecture 10/11 > Flashcards

Lecture 10/11 Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

What is a recombinant protein?

A

Proteins derived from the expression of recombinant DNA within living cells

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

Why express and purify recombinant proteins?

A

• Large quantities required for research study in vitro
i.e. for structure determination using X‐ray crystallography or NMR (nuclear magnetic resonance) experiments
• For therapeutics and commercial applications

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

Recombinant protein expression

A

 Bacterial expression systems: regulation using the lac operon
 Insect expression systems
 Yeast & other expression systems

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

Commercially available vectors

A

 pET
 pGEX
 pBAC

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

Fusion proteins: affinity tags (for purification)

A

 6x His tag
 GST
 MBP

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

REQUIREMENTS OF A BACTERIAL EXPRESSION SYSTEM

A
  1. Expression vector (plasmid)
    Sequences for expression and regulation of expression
  2. Host bacterial cells (often modified)
    Important properties:
    (i) Regulate expression
    (ii) Provide suitable environment for expression and correct folding
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

REQUIREMENTS FOR HIGH LEVEL EXPRESSION

A
  • A strong regulated promoter:
  • able to make large numbers of mRNA transcripts
  • able to produce mRNA transcripts when required
  • Effective translation of proteins from mRNA transcripts
  • Correct processing and folding of the translated proteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Key features required for protein
overexpression in a bacterial
expression system

A

-Bacterial promoter (P)
-operator (O) sequences
-Polylinker with unique sites for several restriction endonucleases (i.e. cloning sites)
-Transcription termination sequence
-Selectable genetic marker (e.g. antibiotic resistance)
-ori
-Gene encoding repressor that binds O and regulates P
In addition, expression
vectors/plasmids also often have a gene that encodes an affinity tag for purification

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

What is a promoter for

A

allow efficient transcription of the inserted gene (drives transcription)

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

What is a polylinker/MCS

A

site of insertion for gene of interest

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

What is a operator

A

permits regulation through a specific repressor that will bind to it

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

steps in Producing a recombinant proteins in a Bacterial Expression System

A

Step 1: Insert gene into bacterial expression plasmid
Ligate ‘gene’ insert into bacterial expression plasmid (at MCS) using DNA ligase
>recombinant vector/plasmid
Step 2: Transform recombinant expression vector into host cells (E. coli)
Step 3: Select for transformed E. coli host cells using selectable antibiotic marker
(DNA sequencing to ensure correct clone)
Step 4: Express protein using inducible promoter
Step 5: Purify protein

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

Lac operon: collection of three genes required for lactose transport and metabolism in E.coli

A 
• Adjacent structural genes: lacZ (β‐galactosidase), lacY (lactose permease) 
and lacA (thiogalactosidase transacetylase)
• Allow for the effective digestion of lactose in vivo
• Lactose permease (cytoplasmic membrane) transports lactose into cell
• β‐galactosidase (cytoplasm) cleaves lactose into glucose and galactose
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Control mechanism in vivo for lac repressor

A

• Lac repressor (regulatory protein) encoded by lacI gene
• lacI gene is constitutively expressed
• In absence of lactose, Lac repressor binds tightly to operator sequence
• Lac repressor interferes with binding of RNAP to promoter
> prevents gene transcription by RNA polymerase

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

How to optimise translation

A

Need effective translation of proteins from mRNA transcripts
• Not all 61 mRNA codons are used equally
• Frequently used codons have large pools of their corresponding tRNAs
• The expression system generates large numbers of the target gene mRNA

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

What may go wrong in translation

A

codon usage in the over‐expressed protein differs significantly from the host cell, problems may arise during protein expression.
- truncated proteins
- mis-incorporation of amino acids (e.g. Lys for ARg at the AGA codon)
-inhibition of cell growth/protein synthesis
the expression system needs to maximise the translation of proteins from this mRNA

17
Q

How to make sure that the expression system is maximising the translation of the protein form the corrrect mRNA

A

Use host strain that expresses genes encoding tRNAs for rare codons. A modified cells
E.coli BL21 strain (DE3) Rosetta : extra copies of genes encoding tRNA
example: Yield of human plasminogen activator protein was increased 10‐fold in E. coli Rosetta BL21
(DE3) strain due to an extra gene encoding tRNA for AGG and AGA (Arg)

18
Q

What problem may face during processing

A
  • Many proteins require the formation of disulphide (S‐S) bonds to fold properly
  • Without S‐S bond, proteins may be degraded or precipitate in the cell
  • Expression of proteins occurs in cytoplasm of E. coli
  • Cytoplasm is a reducing environment that can prevent S‐S bond formation
19
Q

What is the solution to disulphide bonds not folding properly

A

Solution 1: Use E. coli strains engineered with mutations in:
• gor (glutathione reductase)
• trxB (thioredoxin reductase)
These altered E. coli strains can promote disulphide bond formation and correct folding in the cytoplasm

Solution 2: Export proteins to this compartment using a leader sequence to promote S‐S bond formation Leader sequence must be incorporated in the expression vector

20
Q

What is an example of leader sequence for solution 2

A

PelB gene:

encodes periplasmic leader sequence of 22 amino acid residues. Localises to periplasm

21
Q

What is the pET vector

A

It contains a expression feature that increases the control on protein induction. An extra level of protein induction. Pt7: promoter for T7 RNA polymerase. But a genetically modified host cell is needed to contain the T7 RNA polymerase gene as well as its normal indulge-nous polymerase gene (eg of host cell is BL(DE3) Host chromosome

22
Q

How does the IPTG impact a plasmid

A 
Under normal growth: 
• System is repressed by Lac 
repressor protein which is 
transcribed from the lacI gene by host cell RNA polymerase
induction of IPTG

• Lac repressor inactivated on both plasmid and bacterial genomic DNA
• T7 RNA pol is produced by host cell RNA pol (under control of Plac promoter, transcribed from host
DE3 lysogen)
• T7 RNA Polymerase can now transcribe the gene of interest (target gene, downstream of PT7
promoter on pET vector)
 over‐expression of protein from target gene

23
Q

What is another commercially available vector

A

pGEX, Ptac modified promoter to increase binding of RNA polymerase
• System is
repressed by lac repressor
Add IPTG
• Endogenous E. coli RNA polymerase will bind promoter and transcribe downstream genes
• Proteins will be translated using the host cells translation machinery

24
Q

How can the structure of the domain help to facilitate the purification process

A

• Proteins have structural domains: fold independently and have discrete
functions
Can add domains to proteins that can be used to facilitate solubility, folding and enhance purification

The added domains are call affinity tag

25
Q

What are the 3 affinity tags

A

6xHis-tag, interacts with cobalt and nickel ions, MBP (Maltose-binding protein), interacts with maltose (amylose), GST (glutathione-S-transferase) interacts with reduced glutathione.

26
Q

REMOVAL OF AFFINITY TAGS BY PROTEASE DIGESTION

A
  • Protease cleavage sites must be encoded when preparing constructs (expression vector) for expressing proteins
  • Must ensure the recognition site is not present in the expressed protein
27
Q

What are the commonly used proteases for removal of affinity tags

A

Thombin(Thr) that cleaves LVPR/GS
Factor Xa that cleaves I(D)EGR
Enterokinase that cleaves EEEEK
3C human rhino virus protease(preScission) LDVLFQ/GP

28
Q

Properties of Polyhistidine moiety (6x Histidine‐tag)

A

• Rarely effects protein structure
• Binds to a nickel (Ni) column under both native and denaturing conditions
IMAC: Immobilised Metal Affinity Chromatography
• Uses agarose or magnetic beads as supports
• Metal chelators are ligands (e.g. Ni‐nitrilotriacetic acid) to which His‐tag binds
• Wash column extensively
• Elute with [high] imidazole

29
Q

How does the affinity tag Polyhistidine moiety (6x Histidine‐tag) work in steps

A
  1. Induce expression of fusion protein with a 6x His‐Tag (cell‐free extract)
  2. Bind protein to Ni‐NTA column (can occur under native or denaturing conditions)
  3. Wash to ensure removal of unbound/non-specific bound proteins
    -amino acid analogue
    -competes with the polyhistidine tag for binding to the Ni-NTA column
    4A. Elite the His-tagged protein with imidazole ([Hight])
  4. Add protease to cleave His-tag from recombinant protein
    4B. When protein is still bound at column, add protease to release
30
Q

Properties of Glutathione S‐transferase (GST) 26 kDa

A
  • Fusion protein with GST tag binds reduced glutathione (GSH) attached to a mattrix (e.g. agarose)
  • GSH is both the substrate for GST and is used to elute proteins from the GST colume (note: in excess)
  • GST-fusion maintains solubility of proteins

Techniques in biomolecular science (12 decks)

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