Experiment 4: Protein Overexpression, Purification

and Analysis of DHFR

Protein Overexpression?

Making a lot of a particular protein,

for purification and use in

downstream applications

Why Overexpress and Purify a Protein?

•characterize function, activity, structure

•use in assays

•raise antibodies

•lab class project and you want to become a well rounded molecular

biologist

Two Choices for purification

1. Isolate from cells that express the protein

Time: Life Sentence

2. Isolate from heterologous cell type

by overexpression

Time: lab period

Why Heterologous Overexpression?

•proteins with low natural abundance

•Need a lot

•proteins predicted from open reading frames (ORFs)

•site-directed mutagenesis

•protein engineering

Heterologous Expression Systems

•prokaryotic– Escherichia coli

– Lactococcus lactis

– other bacteria

•yeast– Pichia pastoris

– Pichia methanolica

– Saccharomyces cerevisiae

• insect cells– baculovirus

• mammalian cells

• in vitro– wheat germ extract

– Escherichia coli extract

www.biochem.wisc.edu/courses/biochem660/materials/slides/AKF.pdf

Avoid issues with toxicity from protein being expressedControl expression – prevent inclusion bodies from forming

Inducible Expression system for this Lab

Induction in this Lab

• Overnight Express Terrific Broth (TB) medium

• Rich medium with lactose for induction

– Simple induction procedure; inoculate and go

• Optimized for high density growth and protein

expression

soluble

insoluble

Accumulation in soluble fraction is preferred

Accumulation in insoluble fraction may occur

Protein Insolubility

•creates problem in expression and isolation of protein•No way to predict

•Insoluble protein forms aggregates called inclusion bodies

Trouble shoot by varying:

•Temperature

•Inducer concentration

•Expression system (strength of expression or localization of protein)

•Fusion partner

Protein Degradation

• Proteases can lead to loss of protein

• Solution: use protease deficient bacterial strains

• Include protease inhibitors in buffers during purification

Purification of overexpressed protein by affinity tags

How to choose your Affinity Tag

•Cost of resin

•Expression level

•Solubility

•Native versus denaturing elution

•Refolding

For this experiment using Ni-NTA Affinity Tag

High imidazole

Low salt buffers

Low imidazole

Experiment 4

Purify histidine tagged mouse DHFR and test recovery from Ni-NTA column

SDS-PAGE and coomassie brilliant blue stain

& Western blot analysis

soluble

insoluble

1. Save 25 µl soluble

2. Save insoluble pellet

3. Save 25 µl flow through

4. Save 25 µl eluate

Questions?