APPARATUS AND PROCESS FOR THE LARGE
SCALE SEPARATION AND RETRIEVAL OF
PROTEINS FROM CELLULAR EXTRACTS
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to protein separation and, more particularly, relates to an apparatus and process for the large scale separation and retrieval of proteins from a 10 crude mixture of cellular matter.
2. Discussion of the Related Art
It is desirable for a wide variety of diagnostic and research procedures to separate and retrieve proteins from a cellular extract. The most popular technique" heretofore used for such separation is known as continuous vertical gel electrophoresis or simply gel electrophoresis. Gel electrophoresis involves the separation and retrieval of enzymes or other proteins from complex mixtures by means of a differential migration of 20 mixture components through a continuous gel medium under the imposition of an electric current. Examples of devices for performing gel electrophoresis are disclosed in U.S. Pat. No. 3,384,564 to Ornstein et al.; U.S. Pat. No. 3,579,433 to Dahlgren; and U.S. Pat. No. 4,877,510 25 to Chen. Each of these patents discloses a device employing lower and upper reservoirs defining lower and upper buffer chambers containing an ionic buffer solution and a conductive gel, respectively. The gel physically separates the two reservoirs. The upper buffer 30 chamber of the device can be separated from the lower buffer chamber by at least one semipermeable membrane which permits the passage of ions but which prevents the passage of enzymes or other proteins being separated. When an electric current is conducted 35 through such a device via positive and negative electrodes in the lower and upper buffer chambers, proteins migrate downwardly through the gel in the upper buffer chamber at different rates which are dependent upon the conductive properties of the individual prote- 40 ins, thus separating into strata each containing one protein or one group of similar proteins. The thus stratified proteins continue to move downwardly through the gel in the upper buffer chamber until further movement is blocked by the semipermeable membrane, if present. 45 The layers of proteins are then removed one by one, e.g., through pumping, before the next layer reaches the membrane.
Gel electrophoretic devices of the type described above typically can separate and retrieve proteins only 50 on a very small scale and then at only a very slow pace. In fact, the typical device is capable of purifying only a few milligrams of proteins in several days or even weeks when used in conjunction with conventional partial purification procedures. This significantly ham- 55 pers research or other procedures which require the retrieval of large volumes of proteins relatively quickly.
Proteins could not heretofore be retrieved at higher rates without encountering substantial difficulties. For instance, as the size of the electrophoretic apparatus is 60 increased to accommodate greater volumes of cellular extracts, the surface area of the gel in the upper buffer chamber does not increase enough to permit sufficient heat transfer to dissipate satisfactorily the heat which is necessarily generated by the electrophoretic process. 65 Since enzymes and other proteins are heat sensitive, the rate of electrophoresis must accordingly be maintained at or below a level above which the temperature of the
enzymes or other proteins being retrieved may become denatured or otherwise inviable.
This problem can be alleviated to some extent by providing a cooling mechanism for reducing the temperature of the gel in the upper buffer chamber, but devices incorporating such cooling mechanisms are expensive and complex to manufacture and operate. In any event, even if an electrophoretic device were to be dimensioned to enhance heat transfer and/or cooled by a cooling device to facilitate larger scale electrophoresis without overheating the protein containing extract, the purification capacity of such a device would be inherently limited because the purification capacity of the gel is limited. This inherent clarification limitation necessarily delays the purification of proteins from the very crude extracts which serve as the base materials in typical electrophoretic processes.
Electrophoretic devices of the type described above also are operationally limited by the location of their negative electrodes. More specifically, such electrodes are typically immovably mounted within the upper buffer chamber or the associated funnel and thus require that the upper buffer chamber always be filled to a certain minimum level for operation.
OBJECTS AND SUMMARY OF THE
It is therefore an object of the invention to provide a system which is capable of separating and retrieving a relatively large quantity of highly purified proteins from a crude cellular extract in a fraction of the time required for retrieval by conventional gel electrophoretic devices.
In accordance with a first aspect of the invention, this object is achieved by employing in concert 1) a horizontal pretreatment device employing the stepwise separation of a mass of cellular extract into a crude mixture of proteins amenable to further purification, and 2) a vertical electrophoretic device which receives as a base material the mixture produced by the pretreatment device and which employs continuous gel electrophoresis to purify further the proteins but which is capable of operating rapidly on a large scale. The pretreatment device preferably includes a receptacle for storing a conductive buffer solution and having a generally horizontal floor, first and second electrodes positioned in the receptacle, and a plurality of cassettes disposed in the receptacle between the first and second electrodes and extending upwardly from the floor. Each of the cassettes includes first and second semipermeable membranes and a layer of a conductive gel matrix sandwiched between the first and second membranes.
Another object of the invention is to provide an apparatus for continuous vertical gel electrophoresis which is simple and durable in construction and operation, which is capable of rapidly purifying proteins in large quantities, and which can be easily filled and operated with different levels of gel without fear of damaging the negative electrode.
In accordance with another aspect of the invention, this object is achieved by providing an electrophoretic device comprising a lower reservoir defining a lower buffer chamber for storing a first conductive medium, a first electrode in electrical communication with the interior of the lower buffer chamber, an upper reservoir defining an upper buffer champher for storing a second conductive medium, the upper buffer chamber being