{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T18:30:45Z","timestamp":1793298645266,"version":"4.5.0"},"reference-count":41,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Biotechnol"],"published-print":{"date-parts":[[2007,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>C. elegans<\/jats:italic>\n                      . 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\n                      <jats:italic>C. elegans<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/1472-6750-7-45","type":"journal-article","created":{"date-parts":[[2007,8,2]],"date-time":"2007-08-02T14:16:25Z","timestamp":1186064185000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins"],"prefix":"10.1186","volume":"7","author":[{"given":"Yunjia","family":"Chen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shihong","family":"Qiu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chi-Hao","family":"Luan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Luo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2007,7,30]]},"reference":[{"issue":"5515","key":"220_CR1","doi-asserted-by":"publisher","first-page":"187","DOI":"10.1126\/science.292.5515.187a","volume":"292","author":"RF Service","year":"2001","unstructured":"Service RF: Structural biology. 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