Cell-free Bacterial Yeast Insect Mammalian Protein Expression Systems

Cell-free

Bacterial

Yeast

Insect

Mammalian

Protein Expression Systems

Protein Expression in Bacteria

1. Advantages/disadvantages

2. Genetic elements essential for the expression

3. Cloning strategies

4. Overview of the available expression systems and expression strains

5. Design of cloning procedures using the VNTI program

Advantages

• Fast growth• Cheap medium and equipment for growing• Good knowledge of the host

Disadvantages

Limitation for expression of eukaryotic proteins due to:

•different frequencies with which the different codons appear in genes of these organisms

E.g. CGT, CGC, CGG, AGG, AGA, CGA code for arginine, but the last 3 (AGG, AGA, CGA) are rarely used in E. coli and it has low amounts of respective tRNAs.

•differences in post-translational modifications (SS bonds, glycosylation etc)

Disadvantages

Accumulation of lipopolysaccharides (generally referred to as endotoxins) …

Goals

To obtain

as much as possible /good expression+good cell growth

soluble folded protein /reduced aggregation

in a form that is easy to purify /use of secretion and tags

Common problem:

High expression=danger of aggregation, decreased cell growth

Genetic Elements Essential for Expression

RBS, START, and STOP

RBS RBS 5-9 n STARTGAAGGAATTCAGGAGCCCTTCACCATG ... ...

Ribosome Bindind Site (RBS):

START codons:

E. coli uses 77% ATG (AUG), 14% GTG (GUG), 8% TTG (UUG) and a few others

STOP codons:

TAG (UAG), TGA (UGA), TAA (UAA)

Genetic Elements Essential for Expression

Promoters

Host’s promoters2500 in the entire genome of E. coli K12 strain

Most frequently used: Plac / Ptac / Ptrc, PPBAD, rhaPBAD

- Regulation of expression

Promoters from phages T7, T3, SP6, T5, PL

- Highly efficient and specific expression

Plac: Regulation

Plac, Ptac, Ptrc: Characteristics

Level of expression (inductor)

Key features

Plac Low level up to middle (IPTG)

Weak, regulated. Suitable for expression of gene products at very low intracellular level. Comparatively expensive induction.

Ptac

Ptrc

(trp-lac)

Moderately high (IPTG)

High-level, but lower than T7 system. Regulated expression still possible.Comparatively expensive induction. High basal level.

PPBAD: Regulation

PPBAD and RhaPBAD

Level of expression (inductor)

Key features

PPBAD Variable from low to high level

(L-arabinose)

Can fine-tune expression levels in a dose-dependent manner. Tight regulation possible. Low basal level. Inexpensive inducer.

rhaPBAD Variable from low to high level

(L-rhamnose)

Tight regulation. Low basal activity. Relatively expensive inducer.

Phage Promoters

Level of expression (inductor)

Key features

T7

T5

Very high

High

Utilizes T7 RNA polymerase.

Utilizes E. coli RNA polymerase.

PL Moderately high (temperature shift)

Temperature-sensitive host required. Less likelihood of "leaky" un-induced expression. Basal level; high basal level by temperatures below 30°C. No inducer.

Combinations

Genetic Elements Essential for Expression

Replication Origin

Plasmid Replicon Copy Number

pBR322 pMB1 15-20

pUC pUC 500-700

pACYC p15A 18-22

pSC101 pSC101 5

colE1 colE1 15-20

Co-expression from two plasmids

Protein Expression in BacteriaPart2

1. Cloning strategies

2. Overview of the available expression systems and expression strains

3. Design of cloning procedures using the VNTI program

Types of Expression Vectors

1

2

3

Insertion into Transcriptional Vectors

Insertion into Translational Vectors

Cloning Using Restriction EnzymesNcoI

HindIII

Cloning Using A-overhangs

TA-Cloning with Topoisomerase

Directional Cloning

CACC

Gateway Technology

Expression of Fusion ProteinsWe may fuse the target protein with

• various tags to facilitate its purification or detection

HHHHHH-target, epitope-target

• highly soluble proteins to improve solubility and to facilitate purification

Thioredoxin-target, GST-target

• signal peptides or other proteins or domains to promote secretion

SP-target

‘Short’ Fusion Protein Construction ATG CAT CAC CAT CAC CAT CAC

‘Long’ Fusion Protein ConstructionNcoI HindIII

HindIII

PstI

pUC18/19

pUC182686 bp

APr

ALPHA

P(BLA)

P(LAC)

ORI

AvaI (435)

BamHI (430)

EcoRI (451)

HindIII (400)

PstI (416)

SmaI (437)

XmaI (435)

XbaI (424)

ApaLI (178)

ApaLI (1121)

ApaLI (2367)

Transcriptional vector

pTrc99

Translational vector

pQE

Translational vector + CDR

pET

pCR&pEXP

pBAD

Expression strains

# Strain Key features 1 BL21 Deficient in lon and ompT proteases 2 BL21* /STAR #1 + deficient in RNaseE

Improves the stability of mRNA transcripts and increases protein expression yield

3 BL21*(DE3) #1 or 2 + carry T7 polymerase under Plac Enables T7 expression

4 BL21*(DE3)pLysS/E #3 + plasmid pLysS or pLysE expressing T7 lysozyme Reduces basal expression of recombinant genes

5 BL21-AI #1 + carry T7 polymerase under PPBAD (araBAD) Enables T7 expression with tight regulation

6 BL21 CodonPlus-RIL

#1 + Enhances the expression of eukaryotic proteins that contain codons rarely used in E. coli: AGG, AGA, AUA, CUA

7 BL21 trxB #1 + deficient of trxB Facilitates cytoplasmic disulfide bond formation

Expression optimization

To optimase:

Level of inducer (e.g. arabinose)

Time of induction

Temperature of the induction step (popular - 18oC overnight)