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Overview of Cell-Free Systems: To understand how the HIV capsid is assembled in the context of the cell, our group established a cell-free system for studying this process. Cell-free systems utilize cellular extracts to reconstitute protein translation and downstream post-translational events, and can be used for the biochemical dissection of the mechanism underlying such events. In essence, cell-free systems combine the best of two worlds -- they can be readily manipulated because they are in vitro systems, but they also mimic the environment where capsid assembly takes place because they contain all the components of the cytoplasm. Cell-free systems were extensively utilized by Gunter Blobel, winner of the 1999 Nobel Prize in Physiology or Medicine, to demonstrate that signal sequences are responsible for directing nascent secretory and membrane proteins to the endoplasmic reticulum (ER) where they enter the secretory pathway. Blobel's signal hypothesis was first developed in the 1970's using cell-free systems, leading to the identification of the signal recognition particle and its receptor, as well as the ER translocation complex. Decades of study by Blobel's lab as well as other labs revealed that cell-free systems faithfully reconstitute events that occur in cells, but often do so more slowly and less efficiently, thereby allowing intermediates to be trapped. Further study of these intermediates can lead to identification of novel cellular machinery, which can then be further studied in cell-free systems as well as in cells (Lingappa, 1989; Lingappa and Lingappa, 2005).
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