Optimization of Hnox Protein Production in Escherichia Coli

Abstract

Hemeproteins carry a variety of different functions in organisms ranging from steroid biosynthesis to respiration, signaling to drug metabolism. In industry, hemeproteins are used for production of drugs such as: pravastatin for lowering cholesterol, progesterone for hormonal treatment of cancers of uterus and cervix, and cortisone used against allergy and inflammation. Hemeproteins can also be used in drug development and biological remediation. The industrial applications of hemeproteins will expand with development of molecular biology and protein design techniques. One of the obstacles to the widespread use of hemeproteins is difficulties in production of high levels of heme bound protein. This study aims to maximize the amount of heme cofactor bound hemeprotein produced. Here, three important factors affecting hemeprotein production in bacteria are examined: induction by isopropyl β-D-1-thiogalactopyranoside (IPTG), δ-aminolevulinic acid (ALA), precursor for heme biosynthesis, and expression temperature. Effects of these factors on production of thermophilic hemeprotein TtHNOX are investigated. Since ALA is an expensive molecule, optimization of the amount of ALA used is important. The most suitable conditions to produce TtHNOX is at low temperature, 0.5 mM IPTG and 1 mM ALA. This study concludes that ALA concentration and expression temperature are important in production of heme bound hemeproteins.