Membrane-associated Protein Expression

The Seeker is looking for a cloning system to achieve plasma membrane-associated protein expression.
This Challenge requires only a written proposal.

Background & Impact Statement

The Seeker wants to express exogenous nucleotide sequences encoding 50-1,000 amino-acid proteins on the plasma membrane surface of animal cells. The Seeker is not particularly interested in high levels of protein production; what is, however, crucially important for him is achieving the targeted expression on the cell surface, which would allow him being able to work with the cells for subsequent molecular interaction studies.

In the past, the Seeker used a high-expression vector from Invitrogen (pIZT vector containing C-terminal V5 epitope tag) to successfully achieve plasma membrane-bound expression of a known membrane-associated protein, with more than 95% of the expression being associated with the cell surface. However, when partial or full-length sequences of other exogenous proteins expected to be membrane-associated have been expressed using the same vector, intracellular expression was evident, but no measurable expression on the surface of the cells has been detected.

The Seeker is therefore looking for a flexible, adaptable surface expression tool that would allow him to drive the expression of practically any protein to the cell surface with the efficiency at least 90% of total expression. More specifically, the Seeker is interested in a robust vector/expression system that would allow reliable targeting of 50-1,000 amino-acid proteins to the plasma membrane surface of different animal (mammalian and insect) cells and then detecting and quantitating the surface-expressed protein by a selected tag/epitope. The full solution must include (or reference) a routine method of transfecting the animal (mammalian and insect) cell line of choice with the expression vector, including means of estimating transfection efficiency.

It is important to repeat that the Seeker is interested in increased protein expression in the plasma membrane (and, ideally, decreased expression elsewhere in the cell). In relation to this, the Seeker would prefer not to overexpress the protein to the level causing internal inclusions. Equally important, the Seeker is interested in applying the proposed expression system to a wide variety of animal cells lines. The Solvers are therefore expected to clearly explain their choice of promoter selected to drive the protein expression and elaborate on whether the promoter is specific for this particular cell line or has ubiquitous efficiency.

The Seeker will pay special attention to the precise mechanism the Solvers use to target protein expression to the cell surface. This mechanism is expected to utilize all available tools for "membrane-tagging" such as membrane-spanning sequences or other membrane-associated "tags", including, but not limited to, a GPI anchor. However, the solution must not include use of signal peptides or transmembrane regions derived from neuraminidase.

The Seeker will also not accept solutions based on baculovirus expression systems. It is also vitally important that the plasma membrane-associated protein expression is not restricted to using a fetal tissue or embryonic cell lines.

To reiterate, the proposed vector/expression system should meet the following Solution Requirements:

1. The proposed vector/expression system should allow reliable targeting of proteins of 50-1,000 amino-acid length to the plasma membrane surface of cells in different animal (mammalian and insect) cell lines in culture.
2. The proposed vector/expression system should allow detecting and quantitating the surface-expressed protein by a selected tag/epitope with the sensitivity of about 1 ng total surface expression/cell.
3. The proposed vector/expression system should allow for a precise estimate of transfection efficiency.
4. The proposed vector/expression system should articulate clear means of targeting protein expression to the cell surface, for example, by using a membrane-spanning sequence or other membrane-associated "tags", including, but not limited to, a GPI anchor. However, the solution must not include signal peptides or transmembrane regions derived from neuraminidase from any source.
5. The proposed vector/expression system should be applicable for a wide range of animal (mammalian and insect) cells and not restricted to using primary cell lines, fetal tissue or embryonic cell lines.
6. The proposed vector/expression system should not be toxic to host cell lines, or otherwise interfere with organelles and/or surface membrane structure and function.
7. The proposed vector/expression system should be simple enough to be used in a conventional molecular biology/cell biology lab. Use of custom-made and/or expensive lab equipment is strongly discouraged.
8. All material and reagents used in the proposed vector/expression system must be safe for human use in the laboratory environment.
9. The proposed method should use only commercially available reagents and equipment.

The Solvers are expected to submit a thorough description of the proposed vector/expression system with an emphasis on how the surface expression will be verified and quantified. A solid scientific rationale explaining why the Solver believes that the proposed system will achieve the objectives of the challenge is a must. Relevant publications have to be provided supporting every aspect of the proposed solution. Of special interest are examples of using the proposed expression system with different sequences and in different cell lines.

The Seeker is looking for a vector/cloning expression system that would allow reliable targeting of proteins of 50-1,000 amino-acid length to the plasma membrane surface of cells in different animal (mammalian and insect) cell lines in culture.

The Solvers are expected to submit a thorough description of the proposed vector/expression system with an emphasis on how the surface expression will be verified and quantified. A solid scientific rationale explaining why the Solver believes that the proposed system will achieve the objectives of the challenge is a must. Relevant publications have to be provided supporting every aspect of the proposed solution. Of special interest are examples of using the proposed expression system with different sequences and in different cell lines.

The Seeker is not looking for a simple review on the subject. The Seeker is looking for solutions that offer the best results and the maximum likelihood for "freedom to practice", i.e. the Solution is relatively unencumbered by patents or patent applications that might prevent the use of the solution. The award is contingent upon theoretical evaluation of the submitted solutions by the Seeker.

Team-based Proposals

We value the diverse nature of the Solvers in our Network, and are now encouraging you to strengthen your Proposals by recruiting team members to work on this Challenge. Past experience shows that collaborating with multi-disciplinary colleagues and submitting Proposals as a team can truly yield great results.

Here’s how to do it.

1. Find team members and have them register for a Solver account on InnoCentive.com
2. Once you have your team, click on the Form A Team tab found in the project Room for this Challenge and fill in the required fields.

That’s it!

All inquiries will be responded to within 1 business day. We look forward to seeing your teams collaborate in our new work environment, and would greatly appreciate any feedback.