Bacterial expression of two human aryl sulfotransferases

Abstract

The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5′ nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs.

Genetically modified Chinese hamster ovary cells for investigating sulfotransferase-mediated cytotoxicity and mutation by 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine

“…Various human and rat STs have been successfully ex pressed in E. coli [Bidwell et al, 1998;] and in Salmonella typhimurium [Glatt et al, 1995]. In the mammalian system, STs have been transiently expressed in COS cells [Coughtrie et al, 1998], and stably expressed in Chinese hamster V79 cells [Czich et al, 1994;Glatt et al, 1998].…”

“…The coding domain of the sulfotransferase in these two cell lines showed 93% homology [Veronese et al, 1994;Bidwell et al, 1998]. The 897-bp PCR-amplified product of the sulfotransferase gene from the genomic DNA of both cell lines is shown in Figure 3.…”

“…Amplification of the sulfotransferase cDNAs from both 5P3H1 and 5P3H3 were carried out using the same set of primers and under the same PCR conditions. The coding domain of the sulfotransferase in these two cell lines showed 93% homology [Veronese et al, 1994;Bidwell et al, 1998]. The 897-bp PCR-amplified product of the sulfotransferase gene from the genomic DNA of both cell lines is shown in Figure 3.…”

Arylsulfate sulfotransferase

“…+ 4-nitrophenol S 4-nitrophenyl sulfate + dopamine <3> (Reversibility: ? <3> [5]) [5] P ? + 4-nitrophenol S 4-nitrophenyl sulfate + enkephalin <1> (Reversibility: ?…”