Gene construction and mutagenesis for site specific modification of protein with carbohydrate.

Abstract

We reported the construction of the structural gene for trans-activator (Tat) protein of human immunodeficiency virus. While maintaining the same amino acid sequence as the viral protein, the corresponding nucleotide sequence was modified to create additional recognition sites for restriction endonucleases and to prevent basepair mismatch during gene assembly. The oligonucleotides were synthesized chemically, purified and assembled into five gene blocks. The gene blocks were cloned into plasmid vectors and later reassembled into a complete gene. The use of gene blocks facilitated in vitro mutagenesis by the cassette mutagenesis method.