The first consideration in protein detection is obtaining an accurate sample to make the measurement. Three questions to ask are:
1) What specific protein are you interested in?
2) What plant tissues is it expressed in?
3) When in a plant’s lifecycle is it expressed?
To answer these questions you will need information about the gene encoding the protein, also referred to as the cloning cassette in biotech labs that produce GMO’s. To understand this we can look back at some basic principles in gene expression. First, one gene code for one protein. Secondly, every gene sequence has three different regions that are each responsible for different aspects of the gene expression process. They are the promoter, coding region, and termination sequence (Fig 2).
The promoter is the part of the gene that the RNA polymerase enzyme first binds to and begins reading the DNA sequence. It is much like capitalizing the first word of each sentence on a page to indicate where an individual sentence begins. The promoter also contains information needed to ‘turn the gene on and off’.
When the gene is turned on, it means the RNA polymerase will proceed to read the DNA sequence and produce RNA. When it is off, no RNA is produced, and therefore no protein can be made. This promoter sequence is also responsible for telling the RNA polymerase how many copies of the RNA molecule to produce. The more RNA copies made, potentially the more copies of the particular protein molecules the cell will produce.
Genetic engineers have used the ability to exchange and alter promoters to develop transgenic plants which produce the protein both where they want it and when they want it. For example, some Bt corn has been transformed with a bacterial gene that codes for the cryIA protein, a protein that is toxic to European corn borer. The cryIA promoter of the Bt bacteria has been replaced by a promoter that allows for expression in plant cells in Bt corn. You have probably noticed that some companies claim their hybrids are resistant for the whole season, while others say resistance will drop off at the end of the season. Also, some companies say that their resistant hybrids produce the protein in all tissues, while others only produce it in specific parts of the plant. These differences occur because some different promoters used by the companies.
Bt expression in all cells: These genetically engineered corn plants have the Bt gene with a promoter from a cauliflower mosaic virus gene encoding the 35S subunit of the ribosome (35S gene) in some genetically engineered plants. The plants will make the Bt protein in all their cells until the plant is dead.
Bt expression in green tissue and pollen: Other genetic engineers have replaced the Bt promoter with a plant gene promoter. Promoters of the phosphoenolpyruvate (PEP) carboxylase gene or a pollen specific gene (PS) have been used to make some recombinant Bt genes. The PEP carboxylase gene is only turned on in green tissue. PS gene expression is pollen specific (as expected). Bt genes with the PEP carboxlase and PS promoters lose expression as the plant senesces. Therefore late season corn borers will not encounter the endotoxin on these plants.
The next DNA region of the gene sequence, the coding region, is directly behind the promoter. Back to our sentence analogy, this is like the actual letters found within a sentence. Their particular order gives each sentence different meaning. The coding region therefore, contains the information needed to build the specific amino acid sequence of a protein. This is the portion of the DNA strand where the structure and function of the protein being produced is determined. For example, a cryIA coding region will code for a slightly different protein than a cry9C coding region, although both are Bt genes.
The final region of the DNA sequence is the termination sequence. This is analogous to the period found at the end of a sentence. This DNA sequence signals to the RNA polymerase enzyme that the gene has been fully transcribed into a strand of RNA. At this point, the RNA polymerase molecule disassociates from the DNA strand and waits in the cytoplasm until the next strand of DNA to be transcribed comes along.
The next phase of gene expression is translating the RNA strand into a sequence of amino acids to form the final protein molecule. This takes place through a series of steps in the cytoplasm. For more details on the translation process, see the online lesson Gene Expression Part 1: Reading Genes to Make Proteins. The final structure and shape of the protein determines its function as an enzyme, structural molecule, or storage capabilities. Understanding a protein’s stability characteristics is important when taking a sample for the protein detection methods. For example, if the protein is not heat-stable, then you may not be able to detect it in processed foods.