The EA1 protease was selected from a panel of over 100 proteases in the ZyGEM culture collection for a number of valuable characteristics that benefited the process of nucleic acid extraction.
The enzyme has a narrow window of actiivty at the relatively high temperature of 75°C, which means it is effectively 'frozen' at ambient temperature, and inactivation is achieved readily above 90°C.
The high temperature of actiivity enhances the proteolytic activity on mesophilic substrates, allowing the use of the protease, to extract DNA or RNA, in PCR and RT-PCR compatible buffers. No chaotropes, salts or pushing the pH up to 10.7! This liberates the researcher from the need for solid phase purification in many applications. It also allows for the detection of lower abundance targets, as the extraction is lossless.
The narrow window of thermal activity allows the EA1 protease to be used in conjunction with mesophilic enzymes, in a single tube extraction. This is illustrated with the prepGEM Bacteria kit, in which lysozyme and the prepGEM EA1 protease are sequentially activated and removed/inactivated with incubations at 37°C, 75°C and 95°C.
When applied to the extraction of total nucleic acid from small cell populations, the EA1 protease - in the RNAGEM formulation - offers unparalled performance by taking advantage of these key characteristics. The high temperature of activity ensures ready cellular degradation, RNAse and DNAse degradation and, importantly, the removal of protein material from the RNA templates released, ensuring maximum reverse transcriptase processivity.
There are a number of papers and app notes published using the ZyGEM technology, from single cryptosporidium to buccal swabs for forensic profiling. Some of these are in our downloads section, but the peer review material is best found here