Proteins work their way into the middle of a polymerase chain reaction
Scientists have so far enjoyed only limited success in applying the polymerase chain reaction (PCR), a highly sensitive method for detecting and measuring DNA, to protein analysis. But now a team of researchers from the Molecular Sciences Institute (MSI), Berkeley, US, has succeeded by taking advantage of an unusual polypeptide known as an intein.
PCR involves repeatedly amplifying DNA strands until they can be detected and measured. The technique could also be applied to proteins, and should be far more sensitive and flexible than existing techniques for protein analysis, such as enzyme-linked immunosorbent assays (ELISAs), which use specific antibodies to identify proteins in a complex mixture. With a PCR-based technique, the identification could be achieved by amplifying and counting DNA labels that are attached to specific proteins. The problem has been in developing an effective and accurate way to attach a protein to a strand of DNA.
The MSI researchers solved this problem with an intein, which is a polypeptide that is expressed within the bulk of another protein. Once that protein has been expressed, the intein cuts itself free and then joins the original protein back together again using a reactive thioester. By attaching an intein to a protein fragment, the researchers could create a sort of self-adhesive fusion protein. They mixed this fusion protein with specially-designed DNA strands, and then added a reducing agent and thiophenol to induce the intein to cut itself free from the protein fragment. This left the fragment with a ’sticky end’ consisting of a reactive thioester; a DNA strand would then attach to this end to form what the researchers termed a ’tadpole’.
The researchers created tadpoles with a number of different protein heads and showed that they could bind to target compounds with great specificity, allowing the compounds to be detected and measured by amplifying the tadpole’s DNA tails with PCR. They were able to use a tadpole with a streptavidin head to detect as few as 600 biotin molecules in solution, making this technique 109 times more sensitive than ELISAs. They were also able to use tadpoles with fairly simple peptide heads to detect two important cellular proteins and a protein subunit of anthrax toxin at sensitivities equivalent to ELISAs’. But these peptide heads were much easier to produce than the antibodies required for ELISAs.
The researchers envisage that a whole range of different protein heads could be developed for these tadpoles, which could find use in a wide range of applications, including disease diagnosis, environmental monitoring and pathogen detection.
I Burbulis et al, Nat. Methods, 2005, 2, 31