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Published on: January 6, 2017
PCR-based gene synthesis to produce recombinant proteins for crystallization
Damien Marsic1, Ronny C Hughes, Miranda L Byrne-Steele
1ExtremoZyme Inc, HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA. ngj@uah.edu.
BMC Biotechnology
|May 1, 2008
Summary
A novel PCR-based gene synthesis method, SeqTBIO, streamlines protein expression and crystallization. This efficient technique enables rapid gene design, synthesis, and implementation for structural biology research.
Area of Science:
- Structural Biology
- Molecular Biology
- Biochemistry
Background:
- Gene synthesis is crucial for structural biology, aiding protein expression and sequence modification.
- Current gene synthesis methods can be complex and unreproducible, leading to reliance on commercial services.
Purpose of the Study:
- To present a novel, efficient PCR-based gene synthesis method (SeqTBIO).
- To demonstrate the integration of gene synthesis, error correction, and subcloning for protein crystallization.
Main Methods:
- SeqTBIO utilizes sequential DNA assembly for homogeneous product generation without extensive purification.
- In vivo homologous recombination is coupled for efficient subcloning and site-directed mutagenesis.
- The method was applied to synthesize genes for PAZ domain and a PolA deletion mutant.
Main Results:
- The SeqTBIO method successfully assembled coding regions for PAZ and PolA.
- Recombinant PAZ and PolA proteins were overexpressed in E. coli.
- Crystallization of both proteins was achieved, yielding samples suitable for X-ray analysis.
Conclusions:
- The study demonstrates a feasible non-automated gene-to-crystal pipeline.
- This integrated approach facilitates gene design, synthesis, recombinant expression, and protein crystallization.
