GENE SYNTHESIS BY A LCR-BASED APPROACH - HIGH-LEVEL PRODUCTION OF LEPTIN-L54 USING SYNTHETIC GENE IN ESCHERICHIA-COLI

Synthetic genes are very useful in genetic and protein engineering. He re we propose a general method for construction of synthetic genes. Sh ort oligonucleotides are joined through ligase chain reaction (LCR) in high stringency conditions to make ''unit fragments'' which are then fused to form a full-length gene sequence by polymerase chain reaction . The procedure is simple and accurate and does not place constraints on sequence and length. In this report, a recombinant leptin gene was synthesized according to the codon preference of Escherichia coli. Bes ides, a substitution of the only Met at position 54 for Leu and an add ition of a Met at the N-terminus were introduced in the synthetic gene . The gene was cloned in the pQE-31 expression vector and was expresse d in E. coli. A large amount of recombinant leptin containing 6x His t ag was produced and purified by Ni-NTA affinity column. Finally, intac t leptin-L54 was released after removing the tag by CNBr cleavage at t he Met residue. (C) 1998 Academic Press.