PCR-based gene synthesis to produce recombinant proteins for crystallization.
BACKGROUND
Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves.


RESULTS
A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille) domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA). The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis.


CONCLUSIONS
We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.
Marsic
Damien
D
ExtremoZyme Inc, HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA. ngj@uah.edu.
Hughes
Ronny C
RC
Byrne-Steele
Miranda L
ML
Ng
Joseph D
JD
D003460:Crystallization; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D005240:Feasibility Studies; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins