LECTURE 19 - RECOMBINANT PROTEIN/PROTEIN EXPRESSION

why do we often express protein in the lab

because it is usually the best way for us to get our protein of interest

what are uses of expressed proteins

stuctural studies

enzymatic studies

antigen/antibody production

pharmaceutical applications

commercial applications

what are some examples of recombinant proteins

human insulin

growth hormone

DNA polymerases

Hepatitis B virus vaccine

what expression system are available for proteins

bacteria

yeast

insect cells

mammalian cell culture

transgenic animals

what questions do we ask when choosing an expression system

what is the protein size

is the protein glycosylated

what yield is required

are there post translational modifications

how does protein size effect choice of system

we use bacteria for small proteins

we use eukaryotic systems for large proteins

how does protein glycosylation effect system choice

we cannot use bacteria because they do not post translationally modify as required to glycosylate

so we use baculovirus or mammalian systems

how does required yield effect system choice

we can use bacteria as they get high yield at low cost

how do post translational modifications effect system choice

must use yeast, baculovirus or other eukaryotic systems which can carry out the modifications

what are the core features of a plasmid

ORI

selectable marker

inducible promoter

MCS

what are the additional features a plasmid may have

poly A tail

stop codon

tags

describe an E.coli expression system

system for high level expression

contains regulated promoters from lac operon

why are lac operon regulated promoters sometimes problematic

they can be leaky, having constant low level expression

can be an issue if the protein which has been cloned in is toxic, then leakage may kill the E.coli

what is an example of an E.coli expression system

PET vectors

what are PET vectors derived from

PBR322

how do PET vectors work

uses T7 bacteriophage gene 10 to promote high level transcription and translation

T7 RNA polymerase expression induced under lac UV5 promoter and IPTG induction

what are the advantages of a bacterial system

quick growth

high yield

low cost

easy manipulation

best understood system

what are the disadvantages of a bacterial system

no glycosylation

no signal peptide removal

eukaryotic proteins may be toxic

no post translational modifications

cytoplasmic degradation

prefernce for using E.coli codons

explain his tag structure

gold or silver are attached to the nickel complex

nickel interacts with his to produce overall tag

how does cloning in yeast work

DNA cloned into MCS, and it is linearised

linear vector hence contains gene of interest

linear vector has recombinant sites that align

what plate do we grow yeast clones on and why

on a histidine deficient plate

yeast naturally do not produce histidine

gene which is inserted will code for histidine, hence only transformed yeast will grow on the deficient plate

what are the features of a typical mammalian expression system

transient mammalian ORI

mammalian promoter

stable mammalian selectable marker

poly A site

bacterial ORI

bacterial selectable marker

strong promoter

describe what transgenics is

the injection of foreign DNA into the pronuclei of fertilised eggs

explain the steps in transgenics

• fertilised egg is removed from female

• DNA injected into male pro-nucleus

• egg replaced into female

• foreign DNA inserts at random into the chromosome of the male pronucleus

what are the 3 ways we can encourage transfection

liposome mediated transfection

DEAE Dextran

Electroporation

explain liposome mediated transfection

polycationic lipid and neutral lipid used, resulting in a liposome with a positive charge

negative DNA and positive liposome form a stable positive complex that can interact with the cell membrane

explain DEAE Dextran

a carbohydrate complex

positively charged

explain electroporation

sudden electrical discharge induces pores in cells

this increases DNA uptake