The Role of Cell‑Free Rabbit Reticulocyte Expression Systems in Functional Proteomics

Abstract

In the broad area of functional proteomics, that is the global characterization of proteins and their function, cell‐free rabbit reticulocyte lysate (RRL) has been used extensively to elucidate the mechanisms of mammalian translation, cotranslational modifications, post‐translational modifications and translocation of proteins. More recently, RRL has been used as the workhorse for manufacturing the proteins engaged in interaction, selection and protein evolution studies from DNA or mRNA libraries either in microarray, display or in vitro expression cloning (IVEC) technologies. This chapter highlights recent functional proteomics applications that use cell‐free mammalian RRL. Abbreviations CMM, canine microsomal membrane; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum‐associated degradation; GPI, glycosylphosphatidylinositol; IVC, in vitro compart‐ mentalization; IVEC, in vitro expression cloning; PCR, polymerase chain reaction; PTT, protein truncation test; RRL, rabbit reticulocyte lysate; RT‐PCR, reverse transcription‐polymerase chain reaction; SP cells, semipermeabilized cells. HighWire Press is a registered trademark of Stanford University. TnT is a registered trademark of Promega Corporation. Introduction In late 1950s and early 1960s researchers first demonstrated that radioactive amino acids could be incorporated into hemoglobin in cell‐free rabbit reticulocyte lysate (RRL).1,2 Since then RRL has been used to elucidate the highly complex events that encompass translation, from initiation to termination.3‐5 RRL has also proven useful in understanding cotranslational folding of nascent polypeptide chains, protein targeting and post‐translational folding. During the 1970s, researchers showed that RRL could be manipulated for exogenously directed mRNA protein synthesis, so that only a single protein of interest was synthesized.6 In the 1990s, the development of coupled transcription/translation, in which RRL is supplemented with T7, T3, or SP6 RNA polymerases further simplified the expression of protein targets.7,8 DNA‐directed protein synthesis in RRL has some advantages over mRNA‐primed RRL including the elimination of mRNA handling, and it usually achieves higher levels of protein synthesis. An advantage of cell‐free RRL over other cell‐free systems (E. coli or wheat germ extracts) is that the mammalian environment more Cell-Free Expression closely mimics human cells. Cell‐free RRL is generated from lysed reticulocytes isolated from phenylhydrazine‐treated rabbits. The lysed reticulocytes are treated with microccocal nuclease to remove endogenous mRNA. The RRL is optimized and supplemented with components that give optimal translation when priming with mRNA and, in the case of a coupled system, optimal translation/transcription for priming with DNA that contains the appropriate RNA phage poly‐ merase promoter sequences. Cell‐free RRL has the same advantages as all cell‐free systems over cell‐based expression sys‐ tems: substantial time‐savings (two hours versus 24–48 hours for protein expression), the ability to adapt to high‐throughput formats, increased tolerance to additives and less sensitivity to toxic or proteolytic proteins. The current use of cell‐free RRL is substantial as illustrated by a HighWire Press® search covering January 2000 to April 2006 that yielded more than 3,000 articles containing the phrase “rabbit reticulocyte.” This chapter will focus on recent applications that use RRL for cell‐free functional proteomics.