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. 2009 Nov 15;394(2):275-80.
doi: 10.1016/j.ab.2009.07.033. Epub 2009 Jul 28.

Profiling of gene-dependent translational progress in cell-free protein synthesis by real-space imaging

Eriko Mikamo-Satoh  1 , Akihiko Takagi, Hiroyuki Tanaka, Takuya Matsumoto, Tsutomu Nishihara, Tomoji Kawai
Affiliations

Profiling of gene-dependent translational progress in cell-free protein synthesis by real-space imaging

Eriko Mikamo-Satoh et al. Anal Biochem. .

Abstract

In general, gene-dependent translational progress affects the efficiency of protein expression. To evaluate the translational progress of protein synthesis, it is necessary to trace the time course of translation as well as the quantity of products. Here we present a new method for tracking translation steps in cell-free protein synthesis using atomic force microscopy (AFM). The cell-free protein synthesis system is useful to track the inherent translational progress of a target gene, whereas conventional UV absorption measurement coupled with density gradient fractionation is difficult to analyze such small sample quantities. Because the high resolution of AFM enables us to clearly count the number of ribosomes included in polysomes, polysome profiles can be obtained directly without complicated fractionation. With this method, we could elucidate the detailed polysome profile with only 1 microl of sample solution. We observed the translational progress of green fluorescent protein synthesis, a model of high-expression protein, as well as human retinoid X receptor. Detailed polysome profiles showed different patterns of translational progress and were clearly associated with the results of time-dependent protein expression. Our study suggests the possibility for comprehensive character analysis of inherent gene-dependent translational progress.

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