Pressure Cycling Technology (PCT) Applications in Extraction of Biomolecules from Challenging Biological Samples
High Pressure Bioscience and Biotechnology
The Barocycler TM NEP3229, a commercially available pressure cycling instrument from Pressure BioSciences, can be used to process up to 0.5 g solid or 1.5 mL liquid samples in a single-use PULSE TM Tube. The Barocycler can rapidly generate alternating hydrostatic pressure between ambient and 235 MPa within a 3 second rise time and millisecond pressure drop time. It was found that, during an exposure to multiple cycles of pressure, biomolecules, such as nucleic acids, proteins, lipids and small
... , lipids and small molecules can be extracted into a lysis buffer from cells and tissues. The composition of the lysis buffer, pressure cycling parameters, and process temperature (4-50ºC) conditions can be adjusted for specific applications. Here, we describe our recent developments in the extraction of biomolecules from important yet recalcitrant biological samples. In an effort of extracting proteins for proteomic studies, samples of microorganisms (Escherichia coli, Saccharomyces cerevisiae, Vibrio parahaemolyticus, Rhodopseudomonas palustris), nematodes (Caenorhabditis elegans), plant (Strelitzia reginae) and animal tissues such as whole Danio rerio (zebra fish), rat liver, brain and adipose tissue, were processed using PCT. 2D gel electrophoresis, liquid chromatography and mass spectrometry were employed in the analysis of the PCT-extracted protein species. It was shown that extracts obtained by PCT frequently yield more protein species, particularly among low abundance, high molecular weight, hydrophobic and basic proteins, than many conventional extraction techniques. PCT can also be beneficial for the extraction and enrichment of nucleic acids from small or difficult to lyse samples. For example, PCT protocols have been developed for DNA extraction from hard-to-break materials, such as bone fragments and teeth, hair and skin. These samples can be in minute quantity, such as single hair or small bloodstain spot on a single cotton thread. In the absence of pulverization and substantial de-calcification, DNA molecules were successfully extracted from exhumed ancient bone fragments for sequence analysis. These studies illustrate the capabilities and potential of PCT applications for extraction of various analytes from challenging biological samples leading to new opportunities in drug discovery, diagnostics and biotechnology.