10] Purification of bacterial luciferase by high-performance liquid chromatography

Abstract

This chapter discusses the purification of bacterial luciferase by high-performance liquid chromatography. Large quantities of bacterial luciferase (Lase) can be rapidly prepared by HPLC using three chromatography procedures, which have their respective counterparts in low-pressure liquid chromatography: high-performance size exclusion chromatography (HPSEC), high-performance anion exchange chromatography (HPAEC), and high-performance hydrophobic interaction chromatography (HPHIC). In particular, HPHIC and its low-pressure counterpart are most useful and provide purifications of Lase that equal or exceed those obtained by affinity chromatography on diphenylpropylamine-Sepharose. The major concern in the HPLC of biological extracts is that the columns will become clogged by particulates, which possibly could ruin a costly HPLC column. Particulates are removed from the solvents by filtration using a membrane filter with a pore size of 0.45 μm or less (Millipore or Nucleopore). The filtered solvents are stored covered to prevent contamination by dust or other debris. The HPLC pumps and columns are secondarily protected from particulates by solvent filters placed on the solvent intake lines. It is recommended that all solvents be filtered on a daily basis to remove any bacteria and microcrystals of salts that form in concentrated buffer solutions. The steps in the purification of luciferase includes: (1) luciferase from photobacterium leiognathi DD17, (2) preparative high-performance size exclusion chromatography (HPSEC), (3) preparative HPAEC, (4) preparative HPHIC, and (5) luciferase from an aldehyde-requiring mutant of vibrio harveyi.