Service A: Cloning

Dependent on later applications or functions of/for the protein, the cloning step can require different degrees of complexity.

Referring to your requirements, we can perform all necessary experiments, including gene modification, prior to insertion into the transfer vector. Alternatively, we support you by advice finding the approriate transfer vector and the optimal conditions to virtually ensure an efficient gene expression for your gene of interest.

With respect to the purification procedure we recommend to introduce a “tag” at the N- or C- terminal end of the recombinant protein from insect cells.

If the recombinant protein is used mainly for in-vitro experiments (e.g. cell culture studies) we recommend for example a “His- tag” (6xHistidin) or a “Strep- tag II” (8 amino acids) . The “tag” is usually 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 [find more detailed infomation at www.iba-go.de].

If the recombinant protein is needed for animal studies (e.g. in mouse or rat) we recommend the usage 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 offers human IgG1-Fc, mouse IgG2b-Fc or rat IgG2a-Fc fragments.

 

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A1) Gene cloning into a baculovirus transfer vector

Material we need from you:

10-30 µg purified and characterized plasmid cDNA bearing the gene to be expressed and containing the appropriate restriction sites for subcloning.

Our service comprises:

Subcloning of the cDNA into the baculovirus transfer vector pBacPAK -8/-9 or into a transfer vector chosen by the customer.

Note: If there are no appropriate restrictions sites, the cDNA first has to be subcloned into another expression vector, e.g. pCR2.1, before cloning into the baculovirus transfer vector!

  • Plasmid-preparation of 10 recombinant clones.
  • Analysis by agarose gel electrophoresis.
  • Mid-scale plasmid preparation of a positive clone.

Results provided to you:

  • a baculovirus transfer vector containing the cDNA of interest.
  • a detailed report sheet.

Expected time range:

1- 4 weeks

A2) Gene cloning into a baculovirus transfer vector including a „tag“

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 with a “tag”.
  • PCR with cDNA template provided by you.
  • Characterisation by agarose gel electrophoresis.
  • Subcloning of the PCR-fragment in the baculovirus transfer vector pBacPAK -8/–9 or in a transfer vector chosen by the customer, containing the “tag” 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 to detect possible mutations!

Results provided to you:

  • A baculovirus transfer vector containing the cDNA of interest.
  • A detailed report sheet.

Expected time range:

4-6 weeks.