title: Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes.
abstract: The assembly of synthetic oligonucleotides into genes and genomes is an important methodology. Several methodologies for such synthesis have been developed, but they have two drawbacks: (1) the processes are slow and (2) the error frequencies are high (typically 1-3 errors/kb of DNA). Thermal damage is a major contributor to biosynthetic errors. In this paper, we elucidate the advantages of rapid gene synthesis by polymerase chain assembly (PCA) when used in combination with smart error control strategies. We used a high-speed thermocycler (PCRJet) to effectively minimize thermal damage and to perform rapid assembly of synthetic oligonucleotides to construct two different genes: endothelial protein C receptor (EPCR) and endothelial cell thrombin receptor, thrombomodulin (TM). First, the intact EPCR gene (EPCR-1, 612 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively. Next, for direct error comparison, another longer gene, the 1548 bp TM gene was constructed from 87 oligonucleotides by both rapid and conventional PCA. The fidelity and accuracy of the synthetic genes generated in this manner were confirmed by sequencing. The combined steps of PCA and DNA amplification are completed in about 10 and 22 min for EPCR-1, 2 and TM genes, respectively with comparable low errors in the DNA sequence. Furthermore, we subcloned synthetic TM, EPCR-1, EPCR-2 and native EPCR-1 (amplified from cDNA) into a Pichia pastoris expression vector to evaluate the expression ability, and to compare them with the native gene. Here, we illustrate that the synthetic genes, assembled by rapid PCA, successfully directed the expression of functional proteins. And, importantly, the synthetic and the native genes expressed proteins with the same efficiency.
authors: Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0643, USA.
authors: Tarlan G
authors: TG
authors: Mamedov
authors: Nisha V
authors: NV
authors: Padhye
authors: Hendrik
authors: H
authors: Viljoen
authors: Anuradha
authors: A
authors: Subramanian
mesh_terms: D001779:Blood Coagulation Factors / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D009154:Mutation / Q000235:genetics; D010843:Pichia; D016133:Polymerase Chain Reaction / Q000379:methods*; D011956:Receptors, Cell Surface / Q000235:genetics*; D018179:Receptors, Thrombin / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature; D013997:Time Factors
chemical_list: D001779:Blood Coagulation Factors; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; C091963:activated protein C receptor