Protein Production in insect cells

Baculovirus Expression Service (BVS)

In analogy to our protein expression services in E. coli we offer also a modular system of services around gene and protein expression in insect cells (especially signal proteins). ReliaTech's “Baculovirus expression service (BVS)” is divided into a series of steps which can be performed either individually or in combination so you can choose the steps you actually need.

These services are designed to adequately cover all specific demands along the line from gene expression to protein purification - starting from gene cloning into the Baculovirus transfer vector over recombinant baculovirus construction, high titer recombinant baculovirus production, recombinant protein bulk production and, finally, protein purification.

All steps are conducted in serum-free medium to ensure safety and to yield a high purity of the recombinant protein.

Along with the regular project updates, you will receive progress reports at the end of each service step. We will guide you from step to step dependent on your authorization for us to proceed.

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.


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

Please, 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!

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

Material we need from you:

  • 10-30 µg purified and characterized transfer vector cDNA bearing the gene of interest. The vector is either provided by you or prepared by us using our Service A.

Our service comprises:

  • Ethanol precipitation of transfer vector DNA for sterilisation and quality control by agarose gel electrophoresis.
  • Cotransfection of insect cells with transfer vector DNA and linear baculogold virus DNA.
  • Plaque purification of six recombinant viruses from cotransfection supernatant.
  • Cell infection of 3x106 insect cells with each recombinant virus and preparation of virus stocks.
  • Cell infection of 3x106 insect cells with each virus stock for preparation of cell supernatant and cell pellet.

Results provided to you:

  • 2 ml of each recombinant virus stock.
  • 2 ml of each supernatant or - as an alternative - 3x106 of each infected cell pellet for your own screening purposes.
  • A detailed report sheet.

Note: If required, we determine the protein expression level by Western blotting for you*.
(* option depends on the availablity of appropriate antibodies)

Expected time range:

7-10 weeks.

Material we need from you:

2 ml of recombinant virus stock, preferably with a known viral titer. The sample is either provided by you or prepared by us if Service B has been performed.

Our service comprises:

  • Infection of 0.75x108 insect cells and harvesting of supernatant at 72 h post- infection (100 ml virus stock!).

Note: Upon customer request, this service can be performed with or without serum!

  • Viral titer determination of the viral stock using plaque assay.

Results provided to you:

  • 100 ml of recombinant virus stock at about 5x107 to 1x108 pfu/ml.
  • A detailed report sheet.

Expected time range:

3–4 weeks.

Material we need from you:

10 ml (for study in flasks) or 100 ml (for study in 500 ml spinners) of high titer stock of a recombinant virus. The sample is either provided by you or prepared by us using Service C.

Our service comprises:

  • Infection with the recombinant virus and control virus of 10 flasks or 2 spinners containing insect cells.
  • Harvesting of of recombinant and control virus samples (cells and supernatant) at 24 h, 48 h, 72 h, 96 h and 120 h post-infection.

Note: This step is optional. Usually, protein yields are best between 72- 96 h post-infection.

Results provided to you:

  • Harvested samples collected from recombinant virus, to determine the relative amounts of the recombinant proteins at different times post - infection.
  • Samples collected from control virus.
  • A detailed report sheet.

Expected time range:

2 - 4 weeks

Material we need from you:

10 ml to 1 L of high titer recombinant virus stock depending on the final production volume needed. The sample is either provided by you or prepared by us if Service C has been demanded.

Our service comprises:

  • Direct infection of insect cells in spinner flasks at the desired volume.
  • Harvesting of the cells and supernatant.

Results provided to you:

  • Cell pellet or supernatant corresponding to the infected culture volume.
  • A detailed report sheet.

Expected time range:

 

The purification of a recombinant protein is the most complex step in the whole service cascade. While cloning of a gene into a transfer vector is often highly dependent on the cDNA fragment size, an efficient purification procedure depends on the biochemical characteristics of the individual protein (e.g. amino acid composition, IP value, glycosylation, stability). In additon individual features of the protein (e.g. secreted, cytoplasmatic or membrane-bound protein, mono- or oligo- or polymeric protein) must be take into account establishing an efficient purification strategy.

Adding a “tag” to a protein (either N- or C- terminally) strongly supports the purification or at least the enrichment of the protein applying standard purification protocols (e.g. using protein A or G Sepharose for the Fc-tag). In addition the broad commercial availability of “tag” - specific antibodies allows an excellent fallback option for protein detection as well as for monitoring all purification steps if antibodies for the specific protein of interest are not available yet.

With step E of our protein production services in insect cells we support you to design and establish the optimal strategy to purify your protein of interest.

We are looking forward to support you by E-mail or Phone to find a solution suitable for you. Please contact our technical staff for more details.

Note: This part is offered only in combination with step E and a recombinant protein containing a “tag”! For purification of recombinant proteins without an established purification protocol the price will depend on the required time and material needed!

Material we need from you:

Cell pellet or supernatant from service E.

Our service comprises:

  • Preparation of supernatants or cell pellets for purification.
  • Preparation of columns and buffers.
  • Protein purification by affinity chromatography.
  • Analysis of all fractions by SDS-PAGE and/or Western blotting using a „target”- or „tag”-specific antibody.
  • Determination of protein purity by SDS-PAGE and subsequent silver staining.

Results provided to you:

  • Total yield of recombinant protein gained from the volume scale requested by you.
  • A detailed report sheet.

Expected time range:

Dependent on the volume ordered.