Bacterial expression of two human aryl sulfotransferases

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Human sulfotransferases (HASTs) are crucial enzymes involved in various biological processes.
• Optimizing heterologous protein expression in microbial systems like E. coli is essential for biochemical and functional studies.
• Codon usage bias can influence mRNA stability and translation efficiency, impacting protein yields.

Purpose of the Study:

• To investigate the impact of codon optimization at the N-terminus of human aryl sulfotransferase 1 and 3 (HAST1 and HAST3) on protein expression and activity.
• To evaluate the suitability of the pKK233-2 E. coli expression vector for producing human sulfotransferase cDNAs.

Main Methods:

• Site-directed mutagenesis was used to replace a single N-terminal codon in HAST1 and HAST3 with an E. coli-preferred codon.
• Polymerase chain reaction (PCR) was employed to introduce the nucleotide substitution while preserving the amino acid sequence.
• Protein production, subcellular localization, and catalytic activity of the modified enzymes were assessed.

Main Results:

• The N-terminal codon modification resulted in minimal changes to protein production levels.
• Subcellular localization of the expressed proteins remained largely unaffected by the codon change.
• Catalytic activity of HAST3 showed no significant alteration after the codon substitution.
• The pKK233-2 E. coli vector demonstrated suboptimal performance for expressing human sulfotransferase cDNAs.

Conclusions:

• A single N-terminal codon change has a negligible effect on HAST1 and HAST3 expression and function.
• The study highlights potential limitations of the pKK233-2 E. coli vector for expressing human sulfotransferases.
• Further optimization strategies may be required for efficient recombinant production of human sulfotransferases in E. coli.