Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins.
BACKGROUND
Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics.


RESULTS
With bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies.


CONCLUSIONS
The DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics.
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. chenyj@uab.edu <chenyj@uab.edu>
Yunjia
Y
Chen
Shihong
S
Qiu
Chi-Hao
CH
Luan
Ming
M
Luo
D000818:Animals; D017173:Caenorhabditis elegans / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D011994:Recombinant Proteins