Efficient translocation and processing with Xenopus egg extracts of proteins synthesized in rabbit reticulocyte lysate

Summary

Cell-free translation/translocation systems are broadly applied to examine gene expression and characterize the structure-function relationship of gene products. We present the characterization of Xenopus egg extract (XEE) translocation and processing of proteins synthesized in rabbit reticulocyte lysate. The XEE was prepared from eggs laid by adult female frogs that received serial injections of gonadotropins. The eggs were then dejellied in 2% l-cysteine-HCl and the cytoplasm extracted by centrifugation at 10,000 rpm for 15 min. The in vitro translocation and processing of XEE was examined with a cell-free translation system containing reticulocyte lysate, and appropriate messenger ribonucleic acid (RNA) or complementary deoxyribonucleic acid plasmids with RNA polymerase. Cell-free production of the following proteins were used to assess posttranslational modifications: Escherichia coli β-lactamase for signal sequence cleavage, Sarcomyces cerevisiae α-mating factor for translocation and N-linked glycosylation, the soluble protein luciferase for functional activity, and the membrane-bound human insulin receptor for translation efficiency. All translation products were identified by [³⁵S]-methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The results demonstrate that (1) XEE produces near-complete signal sequence and N-glycosylation processing of proteins synthesized in reticulocyte lysate, (2) XEE contains endoplasmic reticulum-equivalent microsomes, which allows for protein translocation and protease protection, (3) the addition of XEE in the translation reaction does not affect synthesis and chemiluminescence activity of luciferase, (4) XEE is efficient in processing the nascent 160-kDa human insulin receptor precursor, a transmembrane protein, and (5) as compared to canine pancreatic microsomes, XEE translocation efficiency is minimally decreased with the addition of dimethylsulfoxide. These results are the first description of the combined use of XEE with reticulocyte lysate and clearly demonstrate a higher efficiency of translocation and processing compared to canine pancreatic microsomes. This method of cell-free translation and processing allows for more extensive in vitro examination of posttranslational modifications of secretory and membrane-bound proteins.