Construction of a mutant library of horseradish peroxidase gene by directed evolution and development of an in situ screening method

Abstract

A process of directed evolution applied to obtain a library of mutants of horseradish peroxidase (HRP) enzyme is described. We have introduced slight variations into the original DNA shuffling protocol. A DNA template was prepared by PCR amplification and digested with Dnase I during 1 hour. Dnase I products were concentrated by precipitation with isopropanol. Gel electrophoresis showed fragments of the desired size range (20-600 pb) without a full-length template remaining in the reaction mixture. A high concentration of fragments was crucial for performing PCR without primers. In this case, a template concentration of 32.5 ng/mu l was appropriate. Amplification of recombinant genes in a standard PCR reaction (template dilution 1:100) produced a smear with a low yield for the full-length sequence. A single product of the correct size was obtained by PCR with nested primers separated from the previously used primers by 40 pb. In our laboratory, native HRP has been functionally expressed in a baculovirus expression vector system. The purpose is to develop the screening of the first generation of random mutants in this system. To facilitate detection of those clones that have high peroxidase activity, we developed a rapid method: after five days postinfection agarose plates with six wells were covered with DAB (3,3´-diaminobenzidine) and H2O2. The appearance of brown-black stain allows visualization of up to 100 active clones/well in only 1 min

High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture

“…In this assay, 3.1 × 10 6 Sf-9 cells per well were infected with a low MOI (0.1 pfu per cell) so that only isolated cells got infected. A first agarose overlay was added and, after 5 days at 28 • C, a second agarose overlay containing a peroxidase substrate (DAB) and hydrogen peroxide facilitated the visualisation of the plaques and allowed identification of infected cells expressing high levels of active HRP C with the naked eye [12].…”

Horseradish and soybean peroxidases: comparable tools for alternative niches?

“…Mutations within the 131 active site entrance revealed the key role of Phe 142 in binding aromatic molecules 132 [34], whilst mutations within the proximal region (the area below the heme plane) 133 disclosed the parts played by Phe 179 in aromatic molecule binding [35] and by His 134 170 in heme group anchorage [36]. Table 2 pastoris, Mendive and co-workers utilised a baculovirus expression system [42]. 152…”