Synthetic peptides are often used as immunogens because they are quickly and easily synthesized and are relatively inexpensive. The doewnside is that they have no secondary, tertiary or quaternary structure, which can be critical for antibodies used in flow cytometry (FACS), immunoprecipitation, immunofluorescence or blocking antibodies that need to react with native structures. When a western blotting antibody is desired, peptides are often tried first. Target antigens in western blots are usually denatured and charge neutralized. Peptides immunogens only have primary sequence and no carbohydrate groups, so immunizing animals with them can yield good western blot antibodies.
Peptides for immunizations are generally 15 amino acids in length with a cysteine at one of the ends. This cysteine residue allow for linkage to carrier proteins such as KLH to increase immunogenicity and BSA for screening. We have used peptides between 8-30 amino acids in length and have linked the peptides to ovalbumin or tetanus toxin. The peptide chosen should contain several charged amino acids (4-6) to increase hydrophillicity. However, too many charged amino acids can be deleterious. One should also check that the peptide is specific for the protein target of interest and that it doesn’t cross react with other proteins. This is done by BLAST analysis of protein databanks. We generally have 10 mgs of peptide made with 3 mgs conjugated to KLH and 3 mgs to BSA. We can also have phospho-peptides generated where the phosphorylation site is in the middle of a 15-amino acid peptide.
We can produce recombinant proteins in bacterial, mammalian, or insect cell expression systems. The major advantage of bacterial systems is the speed of production while the major disadvantages are poor solubility and the lack of post translational modifications such as glycosylation.
Production in mammalian systems takes longer but there are less problems with solubility and post translational modifications. Insect cells offer the same advantages and disadvantages as mammalian cells although the glycosylation pattern in insect cells is not as complex as in mammalian cells. For bacterial cells, we tend to tag the proteins with GST, His or Strep tag. However for mammalian or insect systems we use His, Fc-fusion proteins, myc, FLAG or HA tags. The process usually takes between 1-2 months to get enough protein for making and screening monoclonal antibodies. Generally it requires 3 mgs of protein to immunize animals for making monoclonal antibodies. It is important to have a protein for screening with a different tag than for immunizations . Since it is only used in screening, about 1 mg of protein is usually sufficient. We can also produce up to a gram of protein using a Fibercell bioreactor system. This process takes between 2-4 months.
The process of recombinant protein production usually begins with a pilot study to optimize expression conditions and to determine the levels of expression. For bacterial systems, we start by testing 4 different conditions with small cultures for expression and solubility and performing a1-liter prep with the optimal conditions. For mammalian systems we generate a 100 mls of supernatant and do a pilot purification. These pilot studies enable us to make a more precise estimate of the number of preps that will be necessary to make the required amount of protein.