- Find out what steps are required to obtain a fusion protein of our own specification
- Are there specific techniques available to detect the fusion of the protein of interest to our antibody segment?
- Tag (sequence: transmembrane protein, antibody sequence, TAG)
- Screen (use affinity of antibody sequence to select for cells that are expressing it properly)
- Are there tricks out there that the folks who have done this can suggest to ensure proper expression?
- Gene fusion techniques:
- DNA level: using restriction enzyme for gene insertion
- Protein level: cross linking
- ELISA: stands for Enzyme-Linked Immunosorbent Assay. It is a diagnostic test utilizing an enzyme-labeled immunoreactant (antigen or antibody) and an immunosorbent (antigen or antibody bound to a solid support). A positive ELISA is typically confirmed using a Western blot assay. There are two types of ELISA: direct and indirect. There are variations of this test, but the most basic consists of an antibody attached to a solid surface. This antibody has affinity for (will latch on to) the substance of interest, for example, human chorionic gonadotropin (HGC), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked HCG will bind. The substance the enzyme acts on is then added, and the amount of product measured in some way, such as a change in color of the solution. ELISA tests are generally highly sensitive and specific and compare favorably with radioimmune assay (RIA) tests.
These are the initial questions that Natalie answered regarding the details of protein fusion:
- Gene fusions are made with restriction enzymes to move DNA around.
- In the original version of the display system, two yeast genes were artificially put under the control of the galactose promoter. This means that when the cells are removed from glucose and grown in galactose the genes get transcribed a lot lot lot and the proteins get made. The proteins are called Aga1 and Aga2 which are naturally exported by the yeast to the cell surface. Aga1 gets imbedded in the membrane and Aga2 attaches to it. They interact with each other through a pair of disulfide bonds.
- How did the Wittrup group detect fluorescence?
- Both c-myc and HA epitopes are present and flank the scFV that is cloneddownstream of the AGA2 sequence. The epitopes can be detected by indirect immunofluoresce, then microscopy or flow.
- How did you get the sequence of the antibody?
- The fusions are encoded by plasmids that the folks in the Wittrup lab purified and sequenced. This procedure has a turnaround time of days, as opposed to protein sequencing which is non-trivial, is more difficult and because of its less likely chances for success has much longer time scales.
- What difficulties and issues are there when doing a gene fusion?
- You have to be smart when you design them. Keep the coding sequence in the right reading frame. Make sure there are no stop sites created. If you need a promoter, there are upstream elements that may regulate expression. Fusion proteins can be unstable or artificially more stable. They may mis-localize. There is a lot of trial and error involved, but in most cases fusions can be made.