(567q) Rational De-Novo Gene Synthesis Using An Automated PCA/PCR with Immunocapture

Polymerase chain assembly (PCA) is a powerful tool for basic biological research and biotechnology applications. During the last several years, major advances have been made in de novo gene synthesis. However, there is still a need for fast and reproducible methods to automatically purify the synthesized genes. Upon completion of PCA, the subsequent PCR-amplified product mixture still contains undesired shorter DNA fragments that hinder cloning efforts. To avoid tedious gel purification, an automated two-column purification has been developed and used in conjunction with rapid PCA. The system enables fast synthesis and isolation of the full length DNA of interest, important for facile cloning of desired DNA fragments. During the PCR amplification step, forward and reverse primers tagged with iminobiotin and bromodeoxyuridine labels, respectively, were used. The automated purification was then performed on the PCR mixture using two affinity/immunocapture columns in series to isolate only the desired full-length product. The procedure has been applied to the genes with sizes varying from 600bp to 3800bp. Follow-up PCR of the purified product, cloning, and sequencing demonstrated the technique's effectiveness in obtaining the pure full-length gene. The purification has also been performed on other synthesized genes, indicating its utility as a general approach.

In this presentation we will present the utility of the above mentioned approach to synthesize chimeric genes, that code for domanins present of on separate proteins. Domains from the thrombin receptor, thrombomodulin and the protein-C receptor, endothelial protein C receptor were selected for the chimera, chimeric gene was synthesized "de-novo" using the PCA/PCR method, cloned and expressed in Pichia Pastoris expression system. The chimeric protein was purfied and characterized.