Expression of Human Placental Aromatase inSaccharomyces cerevisiae

Abstract

A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.

Grape Seed Extract Is an Aromatase Inhibitor and a Suppressor of Aromatase Expression

“…The translation start site is positioned in exon II and one of the 5 ¶-untranslated exon I's of aromatase mRNA is spliced onto a common splicing junction of exon II, upstream of the translation start site (10)(11)(12). It has been found that the various untranslated exon I's are present at different levels in different aromataseexpressing tissues and cells (13)(14)(15).…”

Structure-function studies of human aromatase by site-directed mutagenesis: kinetic properties of mutants Pro-308----Phe, Tyr-361----Phe, Tyr-361----Leu, and Phe-406----Arg.

“…Four mutants of human aromatase have been expressed in Chinese hamster ovary cells using a stable expression method. The activities of these mutants were determined using [13, (2)(3)(4)(5) and the inhibition ofthe enzyme as part ofa therapeutic approach to this disease make the study of aromatase of paramount importance. This enzyme is also of interest to a number of investigators (e.g., refs.…”

“…Although the human placental aromatase cDNA has been cloned and the complete amino acid sequence of the enzyme has been deduced from the nucleotide sequence (10)(11)(12)(13), the structure-function relationship of the enzyme has yet to be established. A mammalian cell expression plasmid, pH P-A~io, containing the human placenta aromatase cDNA, was constructed recently, and this enzyme was expressed in Chinese hamster ovary (CHO) cells by a stable expression method using this plasmid (14).…”

“…Because aromatase exhibits no more than 20-30o sequence homology with other cytochromes P450 (11,13,16), aromatase represents a unique cytochrome P450 gene family. Through sequence comparison with other cytochromes P450, several homologous regions have been identified and thought to be important for the substrate binding.…”

Extracellular Expression and Catalytic Bioactivity of Optimized Human Aromatase (CYP19) in Pichia Pastoris

“…The expression of gene engineering techniques have been employed for the high throughput aromatase inhibitor screening kit. Although human CYP19 has been expressed in E.coli [5], yeast [6], and insect cells [7][8][9], only inclusion body was obtained from E.coli expression due to the lack of necessary protein modification system. On the other hand, even if it can be actively expressed in eukaryotic cells, such as yeast and insect cells, the harsh cultivation conditions and low growth rate of the insect cells limit its extensive application for producing aromatase.…”