FORMATION OF PROLIFERATE. THESE
HETEROKARYONS, BECOME HYBRID
WHICH ARE CELLS, WHICH ARE THEN CULTURED THEN CLONED
Figure 8–4 The production of hybrid cells. It is possible to fuse one cell with another to form a heterokaryon, a combined cell with two separate nuclei. Typically, a suspension of cells is treated with certain inactivated viruses or with polyethylene glycol, each of which alters the plasma membranes of cells in a way that induces them to fuse. Eventually, a heterokaryon proceeds to mitosis and produces a hybrid cell in which the two separate nuclear envelopes have been disassembled, allowing all the chromosomes to be brought together in a single large nucleus. Such hybrid cells can give rise to immortal hybrid cell lines. If one of the parent cells was from a tumor cell line, the hybrid cell is called a hybridoma.
indefinitely so as to produce that antibody in unlimited quantities. In principle, therefore, a monoclonal antibody can be made against any protein in a biological sample. Once an antibody has been made, it can be used to localize the protein in cells and tissues, to follow its movement, and to purify the protein to study its structure and function.
Tissues can be dissociated into their component cells, from which individual cell types can be purified and used for biochemical analysis or for the establishment of cell cultures. Many animal and plant cells survive and proliferate in a culture dish if they are provided with a suitable culture medium containing nutrients and appropriate signal molecules. Although many animal cells stop dividing after a finite number of cell divisions, cells that have been immortalized through spontaneous mutations or genetic manipulation can be maintained indefinitely as cell lines. Hybridoma cells are widely employed to produce unlimited quantities of uniform monoclonal antibodies, which are used to detect and purify cell proteins, as well as to diagnose and treat diseases.
The challenge of isolating a single type of protein from the thousands of other proteins present in a cell is a formidable one, but must be overcome in order to study protein function in vitro. As we shall see later in this chapter, recombinant DNA technology can enormously simplify this task by “tricking” cells into producing large quantities of a given protein, thereby making its purification much easier. Whether the source of the protein is an engineered cell or a natural tissue, a purification procedure usually starts with subcellular fractionation to reduce the complexity of the material, and is then followed by purification steps of increasing specificity.
To purify a protein, it must first be extracted from inside the cell. Cells can be broken up in various ways: they can be subjected to osmotic shock or ultrasonic vibration, forced through a small orifice, or ground up in a blender. These procedures break many of the membranes of the cell (including the plasma membrane and endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. If carefully carried out, however, the disruption procedures leave organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact. The suspension of cells is thereby reduced to a thick slurry (called a homogenate or extract) that contains a variety of membrane-enclosed organelles, each with a distinctive size, charge, and density. Provided that the homogenization medium has been carefully chosen (by trial and error for each organelle), the various components—including the vesicles derived from the endoplasmic reticulum, called microsomes—retain most of their original biochemical properties.