Bioassay development part 4

Principles of Drug Discovery &

Development

Bioassay development

B19FE – Semester 2

8 Lectures

Dr Colin Rickman

([email protected])

B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 2

Bioassay materials

• The bioassays covered in these lectures fall into two broad categories

based on their biological system:

– In vitro assays

– Cell based assays

• In vitro assays usually rely on a source of pure proteins.

– Affinity measures

– Enzyme kinetic measures

• Cellular assays either use the cells as they are or get the cells to

express certain proteins.

– Radioligand binding assays

– Reporter constructs

– FRET assays

• All of these approaches require the use of molecular biology

techniques.

B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development

Molecular biology• Molecular biology is concerned

with the handling and

manipulation of DNA.

• Standard molecular biology

techniques centre around the

use of plasmid DNA.

• Plasmids all contain certain

basic features.– Origin of replication

– Resistance cassette

– Promoter

– Multiple cloning site

• If the plasmid is to be used to

express a protein in bacteria a

phage promoter will be used.

• If the plasmid is to be used to

express a protein in a

mammalian cell a viral promoter

can be used. 3


Molecular biology – Plasmid preparation

• To make a plasmid which

contains the DNA encoding your

protein of interest you need two

items.

1. Expression plasmids (bacterial

or mammalian) are commercially

available.

2. The DNA encoding the protein

which is normally amplified by

the polymerase chain reaction

(PCR).

• PCR involves the cyclical

melting and replication of the

DNA.

• This is only practical due to the

discovery of thermostable

polymerases including Taq. 4



• To make a plasmid which

contains the DNA encoding your

protein of interest you need two

items.

1. Expression plasmids (bacterial

or mammalian) are commercially

available.

2. The DNA encoding the protein

which is normally amplified by

the polymerase chain reaction

(PCR).

• PCR involves the cyclical

melting and replication of the

DNA.

• This is only practical due to the

discovery of thermostable

polymerases including Taq. 5



6


Molecular biology – Plasmid propagation

• After the PCR product is ligated

in to the plasmid there are only a

small number of complete

plasmids.

• Plasmids routinely contain DNA

encoding antibiotic resistance

proteins.

• Plasmids can be inserted in to

bacteria in a process called

transformation.

• Only bacteria containing the

plasmid are resistant to an

antibiotic.

• Growing the bacteria in media

containing antibiotic results in

millions of bacteria all containing

the plasmid. 7


Molecular biology – Plasmid purification

• The standard technique for

plasmid DNA extraction from

bacteria is alkaline lysis.

• The sodium dodecyl sulphate

(SDS) solubilises the bacterial

cell membranes.

• The alkali denatures the

proteins.

• After neutralisation the DNA is

bound to an ion exchange resin.

• The eluted plasmid DNA can

then be stored/used.

• This process is commonly called

a mini prep (small scale) or a

maxi prep (larger scale).

8


Molecular biology – Sequencing

9



• DNA can be sequenced in a

number of ways.

• However, the most common

technique is to used labelled

terminators.

• These are nucleotides where

the 2’ hydroxyl has been

removed.

• This stops elongation of the

synthesised DNA.

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• DNA can be sequenced in a

number of ways.

• However, the most common

technique is to used labelled

terminators.

• These are nucleotides where

the 2’ hydroxyl has been

removed (ddNTP).

• This stops elongation of the

synthesised DNA.

• Originally four separate

reactions were performed each

containing a spike of a single

ddNTP (i.e. one of A, T, G or C).

• Nowadays all four are added at

once, each with a different

fluorophore.11



12


Protein production – Recombinant protein expression

• The most common way to

produce large amounts of pure

protein for use in bioassays is

using bacteria.

• Bacterial expression plasmids

normally contain a promoter

(phage based) which is inhibited

by the lac repressor (lac I)

13


Protein production – Recombinant protein expression

• When lactose (or an analogue)

is added, this binds to lac I

removing the inhibition and the

proteins downstream are

expressed.

• The analogue used is Isopropyl

β-D-1-thiogalactopyranoside

(abbreviated to IPTG).

• IPTG is used as it is non-

hydrolysable by the bacteria and

so remains at high concentration

throughout the process.

14


Protein production – Recombinant protein purification

• Recombinant proteins are normally purified through the use of an

affinity tag.

• In the vector there was a short stretch of DNA coding for the tag

followed by the DNA coding for the required protein.

• There are no stop codons between these two parts so a single fusion

protein is produced.

• In the example on the previous slide a 6xHis tag is added which can

bind to Ni2+-charged resin.

15


Protein production – Recombinant protein purification

• The use of an affinity tag allows you to selectively pull out your

expressed protein from all of the other bacterial proteins.

• This single step results in a huge improvement in protein purity.

• There is a whole range of different purification tags available.

• Additional steps are normally used to improve the purification further

such as ion exchange and size exclusion chromatography.

16


Protein expression in mammalian cells

• Transfection is the process of

inserting foreign DNA into

Eukaryotic cells.

• Eukaryotic cells have evolved to

be very resistant to the

introduction of DNA– Foreign DNA normally only enters cells

during viral infection.

• There are three main

approaches that can be used to

achieve this.– Chemical transfection

– Lipid-based transfection

– Viral transfection

• The approach ultimately used is

entirely dependent on the cell

type to be transfected.

17


Protein expression in mammalian cells – Chemical

transfection• Chemical transfection

techniques mask the negative

charge on the DNA and try to

give it an overall positive

charge.

• One of the simplest techniques

is to mix the DNA with

phosphate and calcium ions.

• The calcium, DNA and

phosphate mix forms a

precipitate which is taken up by

cells using an unknown

mechanism.

• This technique requires careful

control of the pH and ionic

concentrations.

18


Protein expression in mammalian cells – Lipid-based

transfection

• Lipid-based transfection coats the DNA in a positively charged

liposome.

• This liposome is then taken up by the cell through a process called

endocytosis.

• Once in the cell the DNA is released from endosomal compartments

through an unknown mechanism.

19


Protein expression in mammalian cells – Viral

transfection

• Viral transfection utilises the

ability of viruses to get inside

Eukaryotic cells.

• The genes required to be

expressed are inserted in to the

viral genome using molecular

biology techniques.

• Viral particles are added to the

cell culture and taken up by the

cells.

• This approach is very common

for primary cells for which this

may be the only option.

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Bioassay development part 4

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