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. Here we propose a general method for construction of synthetic genes. Short 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. Besides, a substitution of the only Met at position 54 for Leu and an addition 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 expressed in E. coli. A large amount of recombinant leptin containing 6 x His tag was produced and purified by Ni-NTA affinity column. Finally, intact leptin-L54 was released after removing the tag by CNBr cleavage at the Met residue.
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Department of Medical Research, Veterans General Hospital-Taipei, Taiwan, Republic of China. lcau@isc1.vghtpe.gov.tw
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Yang
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D000818:Animals; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D005813:Genes, Synthetic; D020738:Leptin; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis
D020738:Leptin; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D011088:DNA Ligases