Nucleic Acids Research, 1991, Vol. 19, No. 23 6405-6412
© 1991
MOLECULAR BIOLOGY |
A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs
Biochemistry Department, University of Birmingham PO Box 363, Birmingham, B15 2TT, UK
Received September 18, 1991. Accepted November 11, 1991.
We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgramme per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined.
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