Bacerial Expression Strategy

General Strategy

To speed up protein production, we have adopted a strategy of parallel expression of a protein from a variety of vectors containing different tags and/or fusion partners, and a variety of E. coli host strains. This approach should not only gain us a lot of time but also result in a larger number of successfully expressed proteins.

The expression strategy consists of the following two sets of experiments:

1. The expression of a protein in a basic E. coli host strain from a variety vectors with different tags and/or fusion partners.

Our first screen is to express a protein in BL21 (DE3) from modified pET-vectors with the following a selection of tags and fusion partners:

Tag	fusion partner
N-terminal His6-tag
N-terminal His6-tag	thioredoxin
N-terminal His6-tag	glutathione-S-transferase (GST)
N-terminal His6-tag	maltose binding protein (MBP)
N-terminal His6-tag	disulfide oxidoreductase (DsbA)
N-terminal His6-tag	NusA
C-terminal His6-tag

2. The expression of a protein from a standard vector in a number of different E. coli host strains.

The choice of the host strains depends more on the nature of the heterologous protein. The following considerations should be made:

• If the protein contains a high number of rare E. coli codons, it is worthwhile trying to express it in a strain that co-expresses the tRNAs for these rare codons. There are several strains commercially available:

BL21 (DE3) CodonPlus-RIL	AGG/AGA (arginine), AUA (isoleucine) and CUA (leucine)	Stratagene
BL21 (DE3) CodonPlus-RP	AGG/AGA (arginine) and CCC (proline)	Stratagene
Rosetta or Rosetta (DE3)	AGG/AGA (arginine), CGG (arginine), AUA (isoleucine) CUA (leucine)CCC (proline), and GGA (glycine)	Novagen

• If the protein contains one or more disulfide bonds, proper folding is stimulated in host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available from Novagen:

AD494	mutation in thioredoxin reductase (trxB)
Origami	double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor)

• If the protein is toxic to the cell, expression in a strain containing the pLysS or pLysE vector tightens regulation of expression systems using the T7 promoter. These vectors express lysozyme, which binds to and inactivates T7 RNA polymerase. Strains are commercially available from different manufacturers.
• If the protein is membrane-bound, expression in mutant strains C41 (DE3) and C43 (DE3) could improve expression levels.

Reference: Miroux, B. & Walker, J.E. (1996) J. Mol. Biol. 260, 289-298.


Our first screen is to express a protein from a modified pET-vector with an N-terminal His6-tag in the following host strains:

Host strain

BL21 (DE3)

BL21 (DE3) pLysS

BL21 (DE3) CodonPlus-RIL (-RP) or Rosetta (DE3)

Origami (DE3)

For the rapid screening of expression levels and protein solubility from the different vectors and in the different strains we have developed a small scale method using chromatography on magnetic beads.

The results of the first screen will be a starting point for further experiments in case no satisfactory expression conditions have been found. How to optimise expression levels, improve protein solubility, improve protein stability, and decrease protein toxicity will be discussed in the other chapters in this website.

Expression method

A typical expression experiment consists of the following step:

• Picking of a single colony from a freshly streaked plate of the expression host containing the recombinant vector. When the heterologous protein is toxic for the cells, higher expression levels are obtained by using the so-called "plating" method.
• Growing of a starter culture. Inoculate with the picked colony up to 50 ml of rich medium (such as LB or 2xYT) containing the appropriate antibiotic. When a larger starter culture is required, inoculate 4 ml of rich media with the single colony; grow for 4-8 hours at 37°C; and use this to inoculate the starter culture.

Do not let cultures grow at 37°C overnight! It is better to grow overnight cultures at 30°C or lower. Alternatively, the culture can be incubated at 37°C until the OD600 is approx. 1. Then store the culture at 4°C overnight. The following morning, collect the cells by centrigfugation, resuspend them in fresh medium and use this to inoculate the main culture.

The use of ampicillin requires special care. The selectable marker, b-lactamase, is secreted into the medium where it hydrolysis all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture (which can be tested using a plasmid stability test). Some possible solutions are:

• grow overnight cultures at 30°C or lower.

• spin overnight cultures and resuspend the pellet in fresh medium to remove b-lactamase.

• use the more stable carbenicillin instead of ampicillin.

• Inoculation of the main culture and incubation until OD600 reaches 0.4-1. The optimal OD value depends on the culture method and the medium

• For flask cultures using LB-medium an OD600of 0.6 is recommended. To increase the growth rate, we carry out the cultures at 37°C until the OD for induction is reached. Then the cultures are cooled to the induction temperature in ice-water.

Remark: For good aeration, don't use more medium than 20% of the total flask volume.

Induction of protein expression. Protein expression is induced by the addition of the proper inducer or by changing the growth conditions. From this point on the cells will use most of their resources for the production of the target protein and will not grow much further.

For the most used promoters induction conditions are listed below.

Promoter	induction	typical condition	range
trc (hybrid)	addition of IPTG	0.2 mM	0.05 - 2.0 mM
araBAD	addition of l-arabinose	0.2%	0.002 - 0.4 %
PL	shifting the temperature	from 37 to 42°C
T7-lac operator	addition of IPTG	0.2 mM	0.05 -2.0 mM

After induction the cultures are incubated from 3 hours to overnight depending on the induction temperature. Guide lines are given below.

Incubation temperature	incubation time
15°C	overnight
20°C	overnight
25°C	overnight
30°C	5-6 h
37°C	3-4 h

Harvesting of the cell pellet by centrifugation (20 min at 6000 g). Cell pellets are stored at -20°C

Promoters

Promoter	Source	Regulation	Induction	Level of expression
lac	E. coli	lacI, lacIq	IPTG	low
lacUV5	E. coli	lacI, lacIq	IPTG	low
tac (hybrid)	E. coli	lacI, lacIq	IPTG	moderately high
trc (hybrid)	E. coli	lacI, lacIq	IPTG	moderately high
Psyn (synthetic)	E. coli	lacI, lacIq	IPTG
trp	E. coli		tryptophan starvation
araBAD	E. coli	araC	l-arabinose	variable
lppa	E. coli		IPTG, lactose
lpp-lac (hybrid)	E. coli	lacI	IPTG
phoA	E. coli	phoB (positive) phoR (negative)	phosphate starvation
recA	E. coli	lexA	nalidixic acid
proU	E. coli		osmolarity
cst-1	E. coli		glucose starvation
tetA	E. coli		tetracyclin
cadA	E. coli	cadR	pH
nar	E. coli	fnr	anearobic conditions
pL	l	l cIts857	thermal (shift to 42°C)	moderately high
cspA	E. coli		thermal (shift to below 20°C)
T7	T7	l cIts857	thermal
T7-lac operator	T7	lacIq	IPTG	very high
T3-lac operator	T3	lacIq	IPTG
T5-lac operator	T5	lacI, lacIq	IPTG
T4 gene 32	T4		T4 infection
nprM-lac operator	Bacillus	lacIq	IPTG
VHb	Vitreoscilla		oxygen
Protein A	S. aureus

Promoter	RNA polymerase	Inducer/relative strength	Features	Vectors
lac	E. coli	IPTG/+	repressed by glucose; controlled by LacI	pUC; ^
lacUV5	E. coli	IPTG/++	repressed by glucose; controlled by LacI
tac	E. coli	IPTG/+++	CAP site absent, not affected by glucose well controlled by LacI	pMAL; p8CBDek; pKK223-3
trc	E. coli	IPTG/+++	CAP site absent, not affected by glucose well controlled by LacI	pTrc99a; pPROEX; pKK233-2
T5	E. coli	IPTG/+++	with pQE, need to supply expression of LacI repressor (multicopy pREP4 or a strain carrying lacIq gene)	pQE
araBAD	E. coli	l-arabinose/++	repressed by 0.2% glucose; tunable from 0.001 – 0.2% l-arabinose	pBAD
rhaBAD	E. coli	l-rhamnose/++	repressed by 0.2% glucose; very tunable when vector carries rhaRS genes; inducer range = 10-2000 μM l-rhamnose	pRHA-67
tetA	E. coli	aTc/++	pASK75 carries tetR; DH5αZ1 strain or other tetR strain is recommended	pASK75; pZ series
T7 plain	T7	IPTG/+++	very high basal expression; controlled by T7 lysozyme	pET3, 9, 14, 17, 20, 23
T7-lac	T7	IPTG/+++	high basal expression; controlled by T7 lysozyme	pET21, etc.;

+, ++, +++ relative strength of induction.

^not recommended for membrane protein expression

Vector Components

Selectable marker. In the absence of selective pressure plasmids are lost from the host. Especially in the case of very high copy number plasmids and when plasmid-borne genes are toxic to the host or otherwise significantly reduce its growth rate. The simplest way to address this problem is to express from the same plasmid an antibiotic-resistance marker and supplement the medium with the appropriate antibiotic to kill plasmid-free cells. The most used antibiotics and their effective concentrations are listed in table 1.

The use of ampicillin requires special care. The selectable marker, b-lactamase, is secreted into the medium where it hydrolysis all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture. Some possible solution are:

• grow overnight cultures at 30°C or less.
• spin overnight cultures and resuspend the pellet in fresh medium to remove the produced b-lactamase.
• use the more stable carbenicillin instead of ampicillin.

Regulatory gene (repressor). Many promoters show leakiness in their expression i.e. gene products are expressed at low level before the addition of the inducer. This becomes a problem when the gene product is toxic for the host. This can be prevented by the constitutive expression of a repressor protein.

The lac-derived promoters are especially leaky. These promoters can be controlled by the insertion of a lac-operator sequence downstream the promoter and the expression of the lac-repressor by host strains carrying the lacI^q allele (for medium copy number plasmids) or from the same or a helper plasmid (for higher copy number plasmids). Alternatively, repression can be achieved by the addition of 1% glucose to the culture medium.

Origin of replication. The origin of replication controls the plasmid copy number.

Promoter. The promotor initiates transcription and is positioned 10-100 nucleotides upstream of the ribosome binding site. The ideal promoter exhibits several desirable features:

• It is strong enough to allow product accumulation up to 50% of the total cellular protein.
• It has a low basal expression level (i.e. it is tightly regulated to prevent product toxicity).
• It is easy to induce.

An extensive list of possible promoters is given in table 2. The most used promoters are indicated in red.

Transcription terminator. The transcription terminator reduces unwanted transcription and increases plasmid and mRNA stability.

Shine-Delgarno sequence. The Shine-Dalgarno (SD) sequence is required for translation initiation and is complementary to the 3'-end of the 16S ribosomal RNA. The efficiency of translation initiation at the start codon depends on the actual sequence. The concensus sequence is: 5'-TAAGGAGG-3'. It is positioned 4-14 nucleotides upstream the start codon with the optimal spacing being 8 nucleotides. To avoid formation of secondary structures (which reduces expression levels) this region should be rich in A residues.

Start codon. Initiation point of translation. In E. colithe most used start codon is ATG. GTG is used in 8% of the cases. TTG and TAA are hardly used.

Tags and fusion proteins. N- or C-terminal fusions of heterologous proteins to short peptides (tags) or to other proteins (fusion partners) offer several potential advantages:

• Improved expression. Fusion of the N-terminus of a heterologous protein to the C-terminus of a highly-expressed fusion partner often results in high level expression of the fusion protein.
• Improved solubility. Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
• Improved detection. Fusion of a protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody or a binding protein (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) allows for detection of a protein during expression and purification.
• Improved purification. Simple purification schemes have been developed for proteins fused at either end to tags or proteins which bind specifically to affinity resins (see table 3 and references therein).

Protease cleavage site. Protease cleavage sites are often added to be able to remove a tag or fusion partner from the fusion protein after expression. Most commonly used proteases are listed in table 4. However, cleavage is rarely complete and often additional purification steps are required.

Multiple cloning site. A series of unique restriction sites that enables you to clone your gene of interest into the vector.

Stop codon. Termination of translation. There are 3 possible stop codons but TAA is preferred because it is less prone to read-through than TAG and TGA. The efficiency of termination is increased by using 2 or 3 stop codons in series.