In vitro methods for CFTR biogenesis.

Yoshihiro Matsumura, LeeAnn Rooney, William Skach

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)233-253
Number of pages21
JournalMethods in molecular biology (Clifton, N.J.)
Volume741
DOIs
StatePublished - 2011
Externally publishedYes

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Reticulocytes
3' Untranslated Regions
Rabbits
Cystic Fibrosis Transmembrane Conductance Regulator
Messenger RNA
Cell-Free System
5' Untranslated Regions
Protein Biosynthesis
Microsomes
Ribosomes
Canidae
Cultured Cells
Nucleotides
Epithelial Cells
Pharmacology
Proteins
In Vitro Techniques

ASJC Scopus subject areas

  • Medicine(all)

Cite this

In vitro methods for CFTR biogenesis. / Matsumura, Yoshihiro; Rooney, LeeAnn; Skach, William.

In: Methods in molecular biology (Clifton, N.J.), Vol. 741, 2011, p. 233-253.

Research output: Contribution to journalArticle

Matsumura, Yoshihiro ; Rooney, LeeAnn ; Skach, William. / In vitro methods for CFTR biogenesis. In: Methods in molecular biology (Clifton, N.J.). 2011 ; Vol. 741. pp. 233-253.
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