Protein Expression and Purification Services

Services we offer:

Gene synthesis
If required genes can be synthesized allowing the possibility to optimize codon usage. This can in turn lead to increased expression levels. See the separate leaflet about gene synthesis for more information.

Cloning into expression vector
Genes will be cloned into an expression vector. As standard the vector will contain a purification tag (HIS or strep). Cloning into the customers vector of choice can also be performed allowing the addition of promoters, tags or restriction sites.

Protein Expression
Proteins will be expressed in an E. coli expression strain. The protein expression level will be analyzed by SDS-PAGE or Western Blot. Additionally protein solubility can be determined.

Expression Optimization
Baseclear can analyse and optimize the expression level of the desired protein. This is achieved by analyzing and optimizing a number of different parameters including the choice of host strain, the level of induction and growth conditions.

Protein Purification
Small scale purification can be performed using His-tags to demonstrate both tag and protein functionality. Upon request, the protein purification can be optimized and performed at a scale of 10-20 mg.

Results
As a result of the agreed procedure (standard or custom, see table), we will deliver:
- Gene cloned into an expression vector,
in an E. coli expression strain.
- Small aliquot of tagged purified protein
(provided the expression level is sufficient).
- If protein purification is requested, we can deliver up to 10-20 mg of purified protein.
- Report of the expression analysis and (if performed) the protein purification.

Feasibility study:

This study will determine the level of expression for a protein of interest in a standard vector and under standard growth/expression conditions. The process can be broken into two stages:

1) Cloning of the gene of interest into a suitable expression vector (Cost 450 Euro). Alternatively the customer can send an expression vector in which the gene of interest is cloned and the protein expression level will be analysed.

2) Analysis of the expression level and the solubility of the protein. This is performed by growth and induction of the host strain, analysis of the quantity of protein produced and its solubility by SDS-PAGE and western blot. Additionally if the protein has an attached affinity tag a small-scale test can be performed to determine if purification based on the tag affinity is possible. (Cost 300 Euro) (If expression is not seen a 50% reduction in the cost of expression analysis will be given)

Total cost of feasibility study : 750,- Euro order now

no expression 600,- Euro

Optimisation of expression:

The expression level of the protein will be optimised in a number of stages, each stage is designed to optimise a different factor, each of which is know to influence protein expression level. This process will be tailored specifically for each protein and agreed with the customer before initiation. The factors which will be optimised include the following:

Codon usage and fusion proteins: The codon usage of the gene can be analysed and compared to that of the host strain. If required the codon usage can be optimised for expression in the host by gene synthesis. Additionally the fusion to the gene of interest of a fusion protein know to improve solubility and/or expression level (for example Maltose binding protein or thioredoxin) can also be performed to improve the level of soluble protein production.

Host strain: the expression construct will be introduced into a number of E.coli strains specifically designed for protein expression. The strain giving the best expression level will be chosen for further optimisation

Induction level: The level at which a gene expression is induced and the time period over which it is induced can have considerable impact on the level of protein produced and additionally the solubility and activity of the expressed protein. To determine an optimal induction level a range of inducer concentrations can be tested and the optimal inducer concnertation identified.

Growth conditions: Both the growth temperature and the medium in which the host strain is grown has an influence on protein expression. Here a range of growth temperatures, and a number of growth medium will be tested to determine the best growth conditions for maximum protein production.

Protein purification:

If protein expression is visible we can purify it using affinity columns, dependent on expression levels up to 30 mg of protein can be produced. Additionally we can work with the customer to optimise the buffer and elution conditions to obtain a protein which have the maximum possible activity.