Date Published: February 4, 2011
Publisher: Public Library of Science
Author(s): Andrej Trauner, Mark H. Bennett, Huw D. Williams, Vladimir Uversky. http://doi.org/10.1371/journal.pone.0016273
Abstract: We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology.
Partial Text: The translating bacterial ribosome, comprising more than 50 proteins, 3 ribosomal RNAs, mRNA and tRNA, is among the more complex structures in the prokaryotic cell . Ribosomes are central to cellular function – a fact very clearly illustrated by the number of antibiotics, which target their function. The complexity of their composition, structure and function requires the implementation of a range of analytical techniques and almost all of them rely on the isolation of ribosomes using density gradient centrifugation, which is the gold standard for purifying ribosomes prior to further analyses , , , . However, it is a lengthy and labour intensive procedure. The proteomic study of growth-phase dependent as well as environmental stress induced changes in prokaryotic ribosomes and their associated factors has been hindered by the absence of a fast and efficient purification method. Chromatography has been used in the past in an attempt to accelerate and simplify the isolation process , , , , . While such methods never became widely used, there has been a recent renewal of interest in improving the potential of chromatography for isolating ribosomes . Furthermore, advances in genetic manipulation tools have allowed affinity purification to be applied to ribosome isolation, with good results , , . Each of these approaches has its merits; however the speed of separation is always inherently limited by the architecture of the chromatographic matrix. High backpressures caused by the size of ribosomes severely limit the maximum flow rate that can be attained, thus greatly increasing the overall time taken to obtain ribosomal fractions. There is scope to develop a robust, universal, rapid and easy way to isolate ribosomes using chromatography. Monolith columns are a new class of chromatographic stationary phase, based on a highly cross-linked porous monolithic polymer. Unlike conventional chromatography columns packed with porous particles, the monolithic column is a single piece of porous structure of uninterrupted and interconnected channels. The sample is transported through the column via convection leading to very fast mass transfer between the mobile and stationary phase even for large biomolecules . The absence of matrix packing leads to low backpressures allowing high flow rates to be achieved, leading to rapid separations even for very large biomolecules such as protein complexes, immunoglobulins and viruses , . Consequently, we decided to investigate whether monolithic chromatography would be suitable for rapid purification of bacterial ribosomes, and as we have an interest in the composition of mycobacterial ribosome, we used Mycobacterium smegmatis as the model for these studies.
We were successful in developing a new, very rapid chromatographic method for the purification of ribosomes based on the use of strong anion exchange monolith chromatography. The method successfully purified 30S and 50S ribosomal subunits and could be modified through switching elution buffers to isolate intact 70S ribosomes. Speed of isolation is both important in allowing large numbers of samples to be processed in a short time period and also in minimising the contact between ribosomes and proteases and nucleases in the cell extract. With our method, from loading the cell lysate onto the column to obtaining pure ribosomes takes as little as 5 minutes – compared to 8 hours using sucrose gradient ultracentrifugation. We envisage that this technique could be exploited for compositional analysis, as it would provide an efficient and rapid tool for the isolation of ribosomes from multiple samples. The scope of this approach could be broadened by combining it with chemical cross-linking, as we do not believe that modifications incurred by such procedures would adversely affect the chromatographic separation. Furthermore, ribosomes are being investigated as possible vaccines ,. Our method is well suited for such applications and could significantly increase the throughput of such studies.