Total synthesis of human insulin genes, 104 and 80 base pairs long for the insulin A and B chains were designed for the expression in the bacterial systems, respectively. The synthetic DNA fragments were synthesized through the phosphite method on solid support. The enzymatic joinings of the oligonucleotides were employed to give the synthetic DNA duplexes, followed by gene cloning to the N-terminus of the beta-galactosidase gene in bacteriophage M13 mp19 and plasmid pUC19. In order to simplify the characterization of clones and the identification procedures, a couple of DNA fragments encoding short peptides were inserted at the N-terminus of the genes. The usefulness of such devices in conjunction with the expression level of the insulin genes was discussed. Cloned synthetic DNA fragments were sequenced and known that the sequence and leading frame were correct.
사람 insulin A 와 B 사슬 유전자를 고상에서의 phosphite 화학합성법으로 각각 6개의 조각들로 합성하였다가 효소에 의해서 kination 과 ligation 을 시켜 만들었다. 이들을 M13mp19 RF DNA 와 pUC19 plasmid 에 각각 cloning 하였고 linker 를 합성하여 leading frame 을 맞추었다. 이들의 염기서열을 sequencing 으로 조사하여본 결과 leading frame 과 염기서열이 모두 제대로 되어 있음을 알 수 있었다. 이들로 부터 insulin A 와 B 사슬을 많이 만들고 분해도 적게되며 또 확인과 분리를 쉽게하기 위해서 몇가지 polypeptide linker 를 합성하여 이미 만들어져있는 vector 에 끼워 넣었다.
