EXPRESSION OF RECOMBINANT PROTEIN IN E. coli

- Genetic manipulation is easy, - Its culturing is inexpensive, - Expression is fast, - In many cases, the level of expression is high, thus produces large amounts of a specific pr

EXPRESSION OF

RECOMBINANT PROTEIN IN E coli

EXPRESSION OF RECOMBINANT PROTEIN

Bacterial expression system

Escherichia coli is the most commonly used bacterial expression

system for expression of the heterologous proteins

- Genetic manipulation is easy,

- Its culturing is inexpensive,

- Expression is fast,

- In many cases, the level of expression is high, thus produces large amounts of a specific protein

- Majority of foreign proteins are well tolerated,

- Permits studies of the structure and function of proteins

- Can be useful when proteins are rare cellular components or difficult to isolate

Bacterial expression system

1 What type of protein is expressed ?

2 How to get soluble protein expressed in E coli ?

3 Does protein need post-translational modifications for structure/activity?

4 What is the codon usage in expressed protein?

• Interrupted translation, which leads to a variety of truncated

protein products

• Misincorporation of amino acids For instance, lysine for

arginine as a result of the AGA codon.

This can be detected by mass spectroscopy since it causes a decrease in the molecular mass of the protein of 28 Da

• Inhibition of protein synthesis and cell growth

1 Incapable of producing

eukaryotic post-translation modifications

– glycosylation – phosphorylation

2 Certain proteins can not

fold and form inclusion bodies.

3 Deficient in certain tRNAs

commonly found in eukaryotic genes

Inclusion bodies

Common problems with bacterial expression systems

• Low expression levels :

▪ change promoter

▪ change plasmid

▪ change cell type

▪ add rare tRNAs for rare codons on second plasmid

• Severe protein degradation:

– use proteasome inhibitors and other protease inhibitors

– try induction at lower temperature

• Missing post-translational modification: co-express with kinases, etc

• Glycosylation will not be carried out:

– use yeast or mammalian expression system

• Misfolded protein (inclusion bodies):

– co-express with a chaperone

– try refolding buffers

Codon Plus Cell Lines

Especially useful for

the expression of eukaryotic/human

proteins

Elements of expression vector

Promoter, Repressor, Terminator, Translational initiator, Affinity Tag.

MCS: Multi cloning sites,

Selectable Marker gene: Antibiotic Resistant gene

ORI: Origin Replication

Promoter: The commonly used strong promoters in bacterial expression

vectors are lac, trp, tac, and T7 promoters.

Repressor: It is very helpful if the protein of interest is toxic to the bacteria

By the use of repressor the accumulation of toxic level of a protein is

monitored

Terminator: A transcription terminator and Translational terminator

Translational initiator: ribosome binding site (RBS) consists of

Shine-Dalgarno (SD)

Affinity Tag: use of affinity tags has the following advantages:

· It enhances the efficiency of translation of the target mRNA

· It protects the target protein from proteolytic degradation

· Some of the affinity tags (e.g maltose binding protein or MBP) help

in the solubilization of the target protein, hence target proteins remain in the cytoplasm rather than inclusion bodies

Elements of expression vector

Tac Promoter: a constructed hybrid promoter that contains the −35

sequence of the trp promoter and the −10 sequence of the lacZ promoter.

Vectors with fussion of affinity tag

• Facilitate Expression

• Facilitate Purification

Common Protein Fusion Tags

Improving Soluble Cell Lysis

Improving Soluble Cell Lysis

MBP- maltose binding protein

Protein Expression Cell Lines

• Minimize Proteolysis

• Maximize Expression

• Minimize Leaky Expression

• Facilitate Disulfide Bond Formation

• Facilitate Folding (and thus solubility)

Proteases in E coli

Bacterial Cell Lines

Expression levels of proteins

containing rare codons in E coli

To increase the expression, two main methods are available:

• Site-directed mutagenesis to replace the rare codons by more commonly

used codons for the same residue; e.g the rare argenines codons AGA and AGG by the E coli preferred CGC codon

• Co-expression of the genes which encode rare tRNAs There are several

commercial E coli strains available that encode for a number of the rare

codon genes.

+ Often you will obtain a mixture of full-length protein and truncated

species Providing the protein with a C-terminal tag ( e.g His6-tag) will help you to purify only the full-length protein using affinity chromatography.

+ When both above-mentioned methods fail to increase expression

levels, it is time to change expression system and try to express your protein

in yeast or insect cells In cases where the protein contains many rare E coli

codons it is probably better to immediately start with an eukaryotic system.

Advantageous to co-expression the protein that is expressed in

insoluble aggregates (inclusion bodies) with one or more other

proteins

•The co-expression of proteins that play a role in regulation of

expression, such as T7 lysozyme that is expressed from the pLysS

or pLysE vector Strains containing these vector are commercially

available

•The co-expression of the rare codon tRNAs Two strain expressing

different sets of rare codon tRNAs are commercially available from Stratagene

Co-expression from different vectors To ensure plasmid

stability, the vectors should have: different selectable markers, usually antibiotic resistance markers

different origins of replication Often the copy numbers for the vectors will not

be the same This could affect the expression levels of the proteins Therefore, the genes should be cloned into the different vectors (if possible under the

control of different promoters) in order to optimize the expression levels

Co-expression from one vector The genes are cloned into the same vector and could be expressed from one or more promoters If cloned under the regulation of one promoter (di- or multi-cistronic), the order in which the genes are cloned usually affects the expression levels of the proteins

Therefore, several constructs should be made in which the genes are cloned in

a different order to optimize the expression of the proteins

Co-expression

The pET Expression System

One expression system was developed in 1986 by

W F Studier and B A Moffatt: creating an RNA polymerase expression system which was highly selective for bacteriophage T7 RNA polymerase.

www.novagen.com

BL21 cell lines

Champion pET302/NT-His and pET303/CT-His vectors

ROP ORF: Interacts with the pBR322 origin to

facilitate low-copy replication in E coli

Common Protein Fusion Tags

, 2011

1 Thrombin: recognized Leu-Val-Pro-Arg/Gly-Ser

2 Enterokinase: recognized Asp-Asp-Asp-Asp/Lys

4 TEV protease: recognized

Glu-Asn-Leu-Tyr-Phe-Gln/Gly

5 PreScission Protease is a fusion protein of GST and

HRV 3C protease, recognized Gln/Gly-Pro

Leu-Glu-Val-Leu-Phe-pET vector system

www.novagen.com

signal seq = signal sequence for potential periplasmic localization

I = internal tag N = N-terminal tag C = optional C-terminal tag

protease cleavage sites: T = thrombin; E = enterokinase; X = Factor Xa

LIC = ligation-independent cloning

The pGEX Expression System

(Glutathione S-transferase -GST)

The pGEX Expression System

Thrombin (serine

protease) recognized

(LVPR/GS)

The pGEX Expression System

Factor Xa: A blood

clotting protease

recognized IEGR/

The pGEX Expression System

PreScission Protease

recognized

Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro

The pGEX-6P

Human rhinovirus (HRV) 3C protease is a cysteine protease, commonly referred to as the PreScission SiteTM that

recognizes the cleavage site of

Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro, and cleaves between Gln and Gly.

PreScission Protease is a fusion protein of GST and HRV

3C protease Since the protease is fused to GST, it is easily

removed from cleavage reactions using Glutathione

Sepharose™ 4B

Cleavage of GST tag using

thrombin or Factor Xa

Prepare pGEX Vector

pGEX

Cloning Insert into pGEX Vector

Separation techniques for proteins