Overexpression and purification of recombinant proteins in heter-ologous systems are widely used for protein interaction studies, structure and function determination, and production of biopharmaceuticals. Beside the isolation of genes from cDNA libraries, synthesis of genes by PCR techniques has become a common strategy because of the additional possibility to optimize codon usage for high expression yields in heterol-ogous organisms. A set of overlapping primers will reconstitute the desired gene in a two-step PCR method known as gene assembly or gene synthesis (1,2). However, for genes with multiple identical domains or internal sequence repeats, which have gained increased interest as models for repeat proteins (3,4) and as designed proteins (5), this method of gene synthesis may fail due to mispriming. This drawback might be avoided by synthesis of alternative primers for each domain due to the degenerated genetic code (6), but with increasing numbers of domains, the variability of the oligonucleotides will be exhausted. Alternatively, such genes may be assembled by random ligation of monomeric fragments (6,7), by shotgun ligation of oligonucleotides (7), one by one from the monomeric domain by complex time-consuming directed cloning strategies in the desired orientation using compatible overhangs that cannot be digested after ligation (8) or by seamless cloning (9).
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