Journal of Structural and Functional Genomics

, Volume 8, Issue 4, pp 199–208

Functional protein expression from a DNA based wheat germ cell-free system

  • Kate Qin Zhao
  • Robin Hurst
  • Michael R. Slater
  • Robert F. Bulleit
Article

DOI: 10.1007/s10969-007-9035-2

Cite this article as:
Zhao, K.Q., Hurst, R., Slater, M.R. et al. J Struct Funct Genomics (2007) 8: 199. doi:10.1007/s10969-007-9035-2

Abstract

Wheat germ based eukaryotic cell-free systems have been shown to be applicable for both functional and structural analyses of proteins. However, the existing methods might require specialized instrumentation and/or a separate mRNA synthesis step. We have developed a DNA based, highly productive, coupled transcription/translation wheat germ cell-free system that incorporates the normally separate mRNA synthesis step and does not require specialized instrumentation. Using a small-volume batch reaction with fluorescence labeling, DNA templates predicted to encode proteins could be quickly screened for their ability to direct the expression of proteins of the appropriate size. Protein yield can be increased as much as 2 to 4-fold in this system using a dialysis reaction, reaching ∼200–440 μg/ml in 10–20 h. Furthermore, enzyme activities can be assayed directly in the extract without further purification. Simple purification with affinity tags can be achieved in one-step and with minor modifications, efficient SeMet and [U-15N] labeling of >95% can be accomplished in this system. Thus, this efficient cell-free expression system can facilitate both functional and structural proteomics.

Keywords

Cell-free protein synthesis Coupled transcription/translation Fluorescence protein labeling Functional proteomics Structural proteomics Wheat germ 

Abbreviations

BYDV

Barley Yellow Dwarf Virus

CP

Creatine kinase

FfLuc

Firefly luciferase

FluoroTect

The FluoroTect GreenLys in vitro translation labeling system, a lysine-charged tRNA that is labeled at the ε position of the lysine with the fluorophore BODIPY FL

HQ-tag

HQHQHQHQHQ repeats, a metal affinity tag with similar properties to His-tag

hRL

synthetic renilla luciferase with codon usage similar to humans

HT2

HaloTag

MgGFP

Monster green GFP

ORF

Open reading frame

RLU

Relative light unit

RT

Room temperature

SeMet

Seleno-methionine

TBS

Tris buffered saline

TMR

HaloTag TAMRA fluorescence ligand

UTR

Untranslated region

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Kate Qin Zhao
    • 1
  • Robin Hurst
    • 1
  • Michael R. Slater
    • 1
  • Robert F. Bulleit
    • 1
  1. 1.Promega CorporationMadisonUSA

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