In Vitro Methods for CFTR Biogenesis
Cell-free expression systems provide unique tools for understanding CFTR biogenesis because they reconstitute the cellular folding environment and are readily amenable to biochemical and pharmacological manipulation. The most common system for this purpose is rabbit reticulocyte lysate (RRL), supplemented with either canine pancreatic microsomes or semi-permeabilized cells, which has yielded important insights into the folding of CFTR and its individual domains. A common problem in such studies, however, is that biogenesis of large proteins such as CFTR is often inefficient due to low translation processivity, ribosome stalling, and/or premature termination. The first part of this chapter therefore describes parameters that affect in vitro translation of CFTR in RRL. We have found that CFTR expression is uniquely dependent upon 5′- and 3′-untranslated regions (UTRs) of the mRNA. Full-length CFTR expression can be markedly increased using mRNA lacking a 5′-cap analog (G(5′)ppp(5′)G), whereas the reverse usually holds for smaller proteins and individual CFTR domains. In the context of the full-length mRNA, translation was further stimulated by the presence of a long 3′-UTR. The second part of this chapter describes CFTR translation in lysates derived from cultured mammalian cells including human bronchial epithelial cells. Unfortunately, mammalian cell-derived lysates showed limited ability to sustain full-length CFTR synthesis. However, they provide a unique opportunity to examine specific CFTR domains (i.e., nucleotide-binding domain 1 and transmembrane domain 1) under conditions that more closely resemble the native folding environment.
Key wordsBronchial epithelial cells canine pancreatic microsomes cystic fibrosis transmembrane conductance regulator (CFTR) endoplasmic reticulum (ER) in vitro translation membrane protein molecular chaperone protein folding rabbit reticulocyte lysate (RRL)
We thank Dr. Vladimir V. Zeenko for advice in developing mammalian cell lysates and Zhongying Yang for construction and preparation of plasmids. This work was supported by National Institutes of Health grant DK51818 and the Cystic Fibrosis Foundation Therapeutics (W.R.S.) and the Manpei Suzuki Diabetes Foundation (Y.M.).