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BIOL2112 Genes and Genomes > Protein production > Flashcards

Protein production Flashcards

1
Q

What are fusion tags?

A

independent of host organism

easy protein identification & purification

improve stability & folding

can be added N/C -terminal & in-frame

If at N-terminus, remove ATG

if at C-terminus, remove stop codon

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2
Q

How can you purify tagged proteins?

A

Affinity Chromatography

affinity ligand associated with resin

contaminating proteins wash off

wash off protein using solution of appropriate ligand

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3
Q

What is the affinity column used for Glutathione-S-transferase?

A

Glutathione

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4
Q

What is the affinity column used for Maltose binding protein?

A

Amylose (from starch)

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5
Q

What is the affinity column for Hexa-histidine tag?

A

Metals (Nickel or cobalt)

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6
Q

What is the affinity column for StrepTag II?

A

StrepTactin - engineered bacterial protein

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7
Q

how can fusion tags be associated with protease cleavage sites to allow removal?

A

Recognition sites

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8
Q

What are advantages of E.coli as a host for recombinant expression?

A

simple & rapid

easy to transform

well characterised

range of vectors

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9
Q

What are the disadvantages of E.coli as host for recombinant protein expression?

A

requires cDNA - E.coli doesn’t recognise introns

lacks post-translational processing

protein stability issues

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10
Q

What is the T7 promoter system?

A

2 key elements - incorporated into genome. expression of T7 polymerase is controlled by the lac UV5 promoter

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11
Q

What are some codon usage problems?

A

E.coli tRNA can become depleted

choosing a host strain with additional tRNA genes for rare codons can improve translation

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12
Q

What does glucose depletion result in small quantities of?

A

lac permease - allows small amounts of lactose in

converted to allolactose - induces system, prevents lac repressor binding

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13
Q

What are advantages of expression in fungal cells?

A

flexibility over copy number of the plasmid vector

some eukaryotic post-translational modifications e.g. proteolytic processing

deletions of genes for homologous proteins = functional assays in vivo by complementation

cheap

easy to grow

high yields

produce ion channel protein for structural biology

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14
Q

What are the 2 approaches to driving expression far Sacchromyces cerevisiae?

A

Autonomous replication

Integrative

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15
Q

What facts bout expression in Pichia pastoris?

A

AOX1 promoter - drives pacha cell production of the AOX1 enzyme

yield up to 30% of total cell protein

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16
Q

What are problems with expression in Pichia pastoris?

A

does not support episcopal DNA - requires chromosomal integration

Low transformation efficiency

Problem with hyperglycosylation

cells need to be aerated - often need for oxygen

17
Q

How does the AOX1 promoter in Pichia pastoris work?

A

promoter is repressed by glucose

induced by methanol

with from Glc to MeOH = expression

18
Q

What are the 2 vector integration strategies for Pichia pastoris?

A

integration of AOX1 - alcohol oxidase produced, cells can metabolise methanol

Replacement of AOX1 - chromosomal AOX1 replaced, no AOX1 enzyme, cannot metabolise methanol

19
Q

How are pichia grown in bioreactors?

A

agitator
thick cultures
feed in for air - high dissolved oxygen for alcohol oxidase activity

20
Q

What are the 2 ways higher eukaryotic cells are grown by tissue culture?

A

cell monolayers in flasks

suspension cultures in spinner flasks

21
Q

What are the steps for an expression system in insect cells?

A

insert cDNA for target protein in transfer vector

transfect insect cells - produce recombinant virus containing cDNA

infect cell culture at optimal pointing growth cycle

test cells for expression of protein

22
Q

What are key features of the insect cell expression system?

A

viral polyhedrin promoter

polyhedron promoter is active in late stage infection

Baculovirus can be used with Sf9 and Sf21

23
Q

How is a recombinant baculovirus genome constructed?

A

transfer vector encoding protein COMBINED with baculovirus DNA

done by DOUBLE CROSS-OVER RECOMBINATION or TRANSPOSON-MEDIATED RECOMBINATION

24
Q

How does double cross over recombination occur? (baculovirus)

A

POI is downstream of polyhedrin promoter

vector contains orf603 and orf1629

double recombination between 2 odd sequences - incorporate EXPRESSION CASSETTE WITH POLYHEDRIN PROMOTER

25
Q

How does the bac-to-bac system work? (baculovirus)

A

ampicillin resistance casette

2 transposable element sequences - allow recombination with Bacmid DNA

Backed DNA is in a special E.coli strain

26
Q

What are the steps of seating up the bac-to-bac system?

A

1 - integrate ‘gene of interest’ into the BV genome
2 - recovering recombinant BV genome - get first-round virus
3 - make stocks of recombinant virus producing target protein

27
Q

What complexes allow foreign cDNA into a eukaryotic cell cultured in vitro?

A

As a complex with Ca2+

Complexed with a polycationic carrier

As a DNA/lipid complex

28
Q

How does lipofection take place?

A

liposomes when complexed with DNA can fuse with the plasma membrane

enter by endocytosis

become part of an endosome

lipids broken down, DNAse introduced into cell

29
Q

Advantages of mammalian expression system?

A

reliable expression

can be constitutive or inducible by choice of promoter

host cell can be and easily cultured one

stanly transfected cell line will be permanently capable of expressing the target protein

30
Q

Disadvantages of mammalian expression system?

A

high cost of media - expensive to produce cells

integration occurs randomly with low frequency

need to be screened in case integration site is essential for normal cell function

31
Q

What is the Flp-In system?

A

targeted integration at FRT sites

Host cell also has FRT site

lose and gain resistance genes

expression cassette permanently integrated

32
Q

What does the Tet/on and Tet/off system allow control of?

A

control of transfected gene expression

33
Q

What is Tet-on?

A

engineered transcription activator proteins binds to TRE upstream on cDNA ONLY IN PRESENCE OF DOXYCYCLINE ANTIBIOTIC

34
Q

What’s Tet-off?

A

the binding of antibiotic prevents transactivator binding to the TRE

allows expression of protein supporting tumour growth

35
Q

What is the structure of lentivirus?

A

components packaged

36
Q

What is the Lenti-X plasmid?

A

GOI - downstream of the CMV promoter

lots of elements to do with infectivity and packaging of the virus

37
Q

What is the antivirus life cycle in 293T cells?

A

Lenti-X co-transfected with plasmids encoding viral proteins

proteins produced

core particles assembled - genome encodes POI

recombinant virus buds off

BIOL2112 Genes and Genomes (7 decks)

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