Cell-free extract and glycoprotein synthesis system
AssigneeToshio Hara
InventorsToshio Hara, Hiroshi Tarui
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Abstract
Insect cells are stored in a small gas cylinder, and the small gas cylinder is charged with nitrogen gas to pressurize the cylinder. The charged gas is exhausted at once to crush the cells to provide the objective cell extract with translation activity and glycosylation activity. As this method is gentler than the conventional cell-crushing method employing a homogenizer, in addition to translation factors, factors carrying glycosylation activity can also be recovered. As a result, an in-vitro glycoprotein synthesis system capable of performing translation to post-translation glycosylation can be produced.
Claims23
A cell-free extract having a protein synthesis activity for synthesizing protein from a template nucleic acid, and which is prepared by depressurizing cells under pressure and crushing said cells in an inert gas atmosphere.
A cell-free extract prepared by crushing cells having a protein synthesis activity for synthesizing glycoprotein from a template nucleic acid, and a glycosylation activity for adding a sugar chain to the synthesized protein,
- wherein said cell-free extract is prepared by depressurizing said cells under pressure and crushing said cells in an inert gas atmosphere so as to preserve the protein synthesis activity and glycosylation activity of said cells.
A cell-free glycoprotein synthesis composition including a cell-free extract having protein synthesis activity for synthesizing protein from a template nucleic acid, and a cell-free extract having a protein synthesis activity and a glycosylation activity for adding a sugar chain to the synthesized protein,
- wherein said cell-free glycoprotein synthesis composition is capable of synthesizing glycoprotein from the template nucleic acid.
A cell-free extract manufacturing device for manufacturing a cell-free extract capable of synthesizing protein from a template nucleic acid by crushing cells having a protein synthesis activity, comprising:
- a container for containing said cells;
- a gas supply unit for filling said container with inert gas;
- and a pressure control unit for pressurizing, and thereafter depressurizing, the pressure inside said container so as to enable the crushing of cells inside said container in a state of preserving the protein synthesis activity of said cells.
A nucleic acid inserted into the upstream of an arbitrary structural gene linked expressibly to a promoter and capable of implementing or promoting the expression of said structural gene from said promoter, wherein said nucleic acid is a secretory signal sequence of a chicken lysozyme or a secretory signal sequence of an SF162 type human immunodeficiency virus (HIV) gp120.
Dependencies are inferred from the claim text.