The success of this step is always dependent on the gene which shall be expressed. Taking into account your specific requirements, ReliaTech can perform all essential experiments, including gene modification, prior to insertion into an expression vector.

Alternatively, assistance is provided to the customer allowing him to choose a suitable expression vector and the right conditions to  ensure the optimal gene expression.

Because E. coli strains are not able to cleave the signal peptide of secreted proteins the cDNA has to be modified. Therefore the cDNAs are normally generated by PCR deleting the sequence for the signal peptide and introducing the appropriate restriction sites for cloning (in many E. coli expression vectors: NdeI and BamHI).

Option A1 - Standard Cloning

Material we need from you:

• 10-30 μg of purified and characterized plasmid cDNA bearing the candidate gene.
• The construct has to contain the appropriate restriction sites for subcloning (e.g. NdeI/BamHI, for further information, please inquire).

Our service comprises:

• Subcloning of the cDNA into the E. coli expression vector, e.g. pET-9a, pET-15b, pET-21b or pCytexP3 or into an expression vector chosen by you.
• Plasmid-preparation of 10 recombinant clones.
• Analysis by agarose-gel electrophoresis.
• Mid-scale plasmid preparation of a positive clone.

Please, note: Appropriate restricitons sites are mandatory for using this service option. To make sure using the proper sites either inquire for further specification or use our PCR cloning service (see below).

Results provided to you

• An E. coli expression vector containing the cDNA of interest.
• A detailed report sheet.

Expected time range: 2-4 weeks

Option A2 - PCR Cloning

Material we need from you:

• 10-30 μg purified and characterized plasmid cDNA bearing the gene to be expressed.

Our service comprises:

• Generation of specific oligonucleotide primers containing the appropriate restriction sites for subcloning (for more details click here, please).
• PCR with the customer's cDNA as template.
• Characterization by agarose-gel electrophoresis.
• Subcloning of the PCR-fragment in the E. coli expression vector e.g. pET-9a, pET-15b or pCytexP3 or in an expression vector chosen by the customer.
• Plasmid-preparation of 10 recombinant clones.
• Analysis by agarose gel electrophoresis.
• Mid-scale plasmid preparation of a positive clone.
• Verification by sequencing.

Note: All cDNA’s generated by PCR have to be completely sequenced due to possible mutations!

Results provided to you:

• an E. coli expression vector containing the cDNA of interest.
• A detailed report sheet.

Expected time range: 4 - 6 weeks
 

PCR cloning - Recommendations

• For an optimal performance of a new protein it is recommended to test different E. coli strains and E. coli expression vectors either induced by a temperature shift or by IPTG.

• With respect to the purification procedure we would recommend introducing a “tag” at the N- or C-terminal end of the recombinant protein. There are E. coli expressions vectors available containing different “tags”. If the recombinant protein is used mainly in in vitro experiments (e.g. cell culture studies) we recommend for example a “His-tag” (6x Histidin) or a “Strep-tag II” (8 amino acids) [www.iba-go.de]. The “tag” is normally too small to interfere with the activity of the protein. For “Strep-tag II” the possibility exists to create an authentic protein by cleavage of the tag. If the recombinant protein is needed for animal studies (e.g. mouse, rat) we recommend the use of an “Fc-tag” (about 26 kDa) to increase the stability and half-life of the protein in the circulation. As an “Fc-tag” ReliaTech can offer human IgG1 and the mouse IgG2b fragments.

This step enables you to produce your own recombinant E. coli strain for your individual production purposes.

Material we need from you:

• 10-30 μg purified and characterized E. coli expression vector DNA bearing the gene to be expressed. The vector is either provided by you or prepared by us (for further information concerning our cloning services, please notice Service A).

Note: For the optimal performance of a new protein it is recommended to test the bacterial host by using several different E.coli strains for the analytical production, e.g. BL21 (DE3), BL21 Star (DE3), Rosetta (DE3) and others (please, inquire).

Our service comprises:

• Generation of a recombinant E.coli strain using standard protocols.
• Isolation and cultivation of 6 single clones over night.
• Analytic production (1ml scale)
• SDS-PAGE and subsequent Coomassie stain of total lysate from induced/non-induced samples.

The induction of our standard constructs for protein expression is performed by temperature shift or IPTG.

Expected time range:

1-3 weeks

The efficient production of protein is dependent on suitable production conditions. We help you to improve your production systems by establishing optimal production conditions for your constructs.

Material we need from you:

• Recombinant E. coli strain established according to conditions following the requirements in Service B (for more detailed information, please inquire).

Our service comprises:

• Generation of pre-cultures
• Induction of protein production by different incubation periods, different incubation temperatures and either different concentrations of IPTG or temperature shift (depending on the system).
• Preparation of inclusion bodies (IB) and cytosolic fraction (CF).
• SDS- PAGE and subsequent Coomassie stain of  IBs and CFs  from induced and non-induced samples, respectively.

Results provided to you:

A detailed report sheet.

Expected time range:

1-3 weeks

Material we need from you:

• Recombinant E. coli strain established according to conditions following the requirements in Service B (for more detailed information, please inquire).
• Concentration of  IPTG as determined by you or by using our Service C.
• Time of strain incubation as determined by you or by using our Service C.

Our service comprises:

• Generation of the pre-culture and subsequent induction of protein production
• Culture according to incubation time
• Harvesting of the biomass

Results provided to you:

Harvested biomass.

Expected time range:

1 week

Material we need from you:

• Biomass either provided by you or alternatively prepared by us if using service D.

Our service comprises:
1. Preparation of the „Inclusion Bodies“ (IB)

• Disintegration of bacteria by lyses buffer and sonification .
• Accumulation of „IBs“ by several centrifugation steps .
• Check of „IB- preparation“ by SDS/PAGE and Comassie stain.

2. Solubilisation of „Inclusion Bodies“

• Solubilisation of „IBs“ .
• Check of „IBs“ by SDS/PAGE and Comassie stain

Note:  Further measurements concering the purification depend on the protein of interest and the information available.

The following features influence the subsequent proceeding.

1. existence of an established purification protocol,
2. the protein is fused to a “tag”,
3. the native protein is a dimer, or
4. there has to be established a new protocol.

In case of 3. the next step is the dimerization reaction followed by a gel filtration to separate monomers from dimers.

In case of 4. the process is continued with ion exchange chromatography (e.g. anion/cation).

Results provided to you

• The total yield of purified protein.
• A detailed report sheet.

Expected time range:
Dependent on the protocol used.