Microsomal vesicles are used to study co-translational and initial post-translational processing of proteins. Processing events such as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by the translation of the appropriate mRNA in vitro in the presence of these microsomal membranes. In addition, processing and glycosylation events may be studied by the transcription/translation of the appropriate DNA in the TnT® Lysate Systems when used with Canine Pancreatic Microsomal Membranes. To assure consistent performance with minimal translational inhibition and background, microsomes have been isolated free from contaminating membrane fractions and stripped of endogenous membrane-bound ribosomes and mRNA. Membrane preparations are assayed for both signal peptidase and core glycosylation activities using two different control mRNAs. The two control mRNAs supplied with this system are the precursor for β-lactamase (or ampicillin resistance gene product) from E. coli and the precursor for α-mating factor (or α-factor gene product) from S. cerevisiae.
The Signal Sequence Control mRNA (E. coli β-lactamase) is transcribed by SP6 RNA polymerase from a plasmid bearing the coding region for the E. coli gene encoding the precursor to β-lactamase (the ampicillin resistance gene product). The RNA is synthesized without a cap analog. This control mRNA is used to assay for signal peptidase activity and is supplied with the Canine Pancreatic Microsomal Membranes System.
The Core Glycosylation Control mRNA (S. cerevisiae α-factor) is transcribed by SP6 RNA polymerase from a plasmid bearing the coding region for the S. cerevisiae α-mating factor. The RNA is synthesized without a cap analog. This control mRNA is used to assay for core glycosylation activity and is supplied with the Canine Pancreatic Microsomal Membranes System.
For more information, see the Protein Expression chapter of the Protocols & Applications Guide.
- Examination of signal peptide cleavage, membrane insertion, translocation and core glycosylation.
- Compatible with the TnT® Lysate Systems, Rabbit Reticulocyte Lysate, and Flexi® Lysate.
- Walter, P. and Blobel, G. (1983) Meth. Enzymol. 96, 84–93.