Expression and purification of recombinant HIV-1 BH10 Tat protein
|Luther, Kelvin B.
|Master of Science (M.Sc.)
|The Human Immunodeficiency Virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS). The HIV-1 BH10 genome encodes a two-exon gene for an 86 amino acid viral transactivator protein (Tat) that markedly enhances viral transcription. The objective of the present research was to develop an expression and purification system to produce HIV-1 BH10 Tat1-86 for structural analysis. We first attempted to express Tat protein with a PelB leader sequence for secretion into the periplasmic space of 'E. Coli'. We next expressed Tat with a cleavable 6-histidine purification tag at the amino terminus. The presence of several codons in the HIV-1 BH10 'tat' cDNA that are rarely used in ' E. coli' was investigated as a cause of low levels of protein expression. To this end, a 'tat' cDNA was produced by splicing nucleotides 1-186 of a Tat gene composed of frequently used 'E. coli' codons with nucleotides 187-258 of the HIV-1 BH10 'tat' cDNA. (Abstract shortened by UMI.)
|Expression and purification of recombinant HIV-1 BH10 Tat protein