1. Field of the Invention
The present invention relates generally to the cellular mechanisms and enzymes involved in the glycosylation of proteins manufactured by the cell. More particularly, the present invention involves altering the enzyme production capabilities of a cell in order to produce a soluble glycosyltransferase which is secreted by the cell and recovered for further use.
2. Description of Related Art
This invention was made with government support under Grant Contract Nos. GM-27904 and GM-11557 awarded by the National Institute of Health. The government has certain rights in this invention. The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
Glycosyltransferases are important enzymes which are essential to the cellular synthesis of carbohydrates. The glycosyltransferases and their role in enzyme-catalyzed synthesis of carbohydrates are presently being extensively studied (43,44). These enzymes exhibit high specificity for forming carbohydrate structures of defined sequence. Consequently, purified glycosyltransferases are increasingly used as enzymatic catalysts in carbohydrate synthesis. Application of these enzymes has been limited because of difficulties in isolating and purifying them. As a result, glycosyltransferases are only available in very small amounts and are extremely expensive.
The isolation and purification of glycosyltransferases is difficult because of their low abundance in cells and because the enzymes are membrane-bound glycoproteins which reside in the Golgi apparatus of cells. Accordingly, the presently utilized purification procedures involve the use of animal tissues from which the enzymes must be extracted and purified. The standard purification techniques, moreover, typically produce preparations which contain a small but significant percentage of the isolated enzymes which remain membrane bound. Thus, these purification procedures are not amenable to large scale production and therefore are not well suited to meet the present and future demands for purified enzymes to be used in research and in industrial applications involving synthesis of carbohydrates.
As a result, there is presently a need to provide methods for producing increased amounts of purified soluble glycosyltransferases, uncontaminated by membrane bound glycosyltransferases, wherein the method is amenable to relatively large scale production of purified enzymes.