Protein production in E. coli

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.