Cloning full-length protein coding sequence from tissues by reverse transcriptase-PCR (RT-PCR) is a routine procedure. However, there are many proteins that have transcript variants due to mRNA alternative splicing. Furthermore, clones must be verified for their full-length expression with time-consuming and labor-intensive procedures, such as DNA sequencing and expression in cultured cells.
Solution
This University of Miami technology was developed for rapid cloning of full-length open reading frame (ORF) cDNA sequences. A phage display vector has been engineered, so that when ORF cDNA sequence is inserted into the vector, a C-terminal biotinylation tag will be expressed, biotinylated and displayed on phage surface. On the other hand, if an out-of-frame cDNA sequence is inserted into the phage vector, no biotin will be displayed on phage surface. The surface display of biotin will allow rapid cloning of full-length ORF cDNA sequences for individual proteins from tissues.
Competitive Advantage
Compared with commercially available cloning kits, this technology has the following advantages:
oHigh ligation efficiency,
oGuaranteed cloning of full-length ORF cDNA sequences with streptavidin enrichment
oRapid re-verification for ORF cDNA sequences by streptavidin binding assay.
oDoes not need expression plasmid with a C-terminal peptide tag, such as FLAG tag or c-Myc tag.
Applications
To clone full-length ORF cDNA sequences
Patent Status
US Provisional Application No.: 61/096,015
Filing Date: September 11, 2008
Licensing Opportunity
The University of Miami is seeking collaborative research and licensing options
About the Inventors
Dr. Wei Li was recruited from Tufts University to become an Assistant Professor at the University of Miami. Trained at University of Nebraska, Yale University and Tufts University, he became an expert in phage display with open-reading-frame (ORF) cDNA libraries. His ultimate goal is to develop ORF phage display as a convenient, efficient and sensitive approach to study protein-protein interaction.
Dr. Xiao Jiang was recruited from Sun Yat-Sen University from China to become a Postdoctoral Associate at the University of Miami. Trained at Sun Yat-Sen University (China) and Neptunus Bioengineering Holdings Co. Ltd. (China), he is an expert in molecular biology and protein biochemistry.
