Hemagglutinin from Echinacea purpurea L. root separation and identification

Abstract

Lectins or hemagglutinins are non-immune origin glycoproteins, which can bind carbohydrate structures in specific and reversible manner. They have big potential for their therapeutical applications such as immunomodulatory, anticancer antibacterial and other activities. Hemagglutinins from Echinarea purpurea L. (Moench) roots haven’t been investigated. The aim of the experiment: To purify and identify hemagglutinins from Echinarea purpurea L. (Moench) roots. Experiment tasks: 1. Purify hemagglutinins from purple coneflower roots; 2. Identify hemagglutinating glycoproteins. Materials and methods: 1. Affinity column with immobilized D-(+)-mannose ligands was equilibrated and unbound proteins were washed out with phosphate buffer saline (PBS) pH 7,4. Hemagglutinins were eluted out of the column with 0.2 M lactose solution in PBS. Following hemagglutinin fraction was collected and checked for hemagglutinating activity. 2. Hemagglutinating active fraction was separated by polyacrylamide gel electrophoresis and immunoblotted for glycosylated proteins. 3. Glycosylated protein bands were identified by liquid chromatography tandem mass spectrometry by sunflower genome database search. Results: 1. Hemagglutinating active proteins from purple coneflower root were separated from non-active proteins and collected into one fraction. 2. Lysin motive (LysM) peptidoglycan binding domain was identified after database search with identity score equal 182.0. Conclusions: 1. Affinity chromatography method was suitable for separation of hemagglutinating active proteins from purple coneflower root crude protein extract. 2. Identified glycoprotein – LysM domain was responsible for the hemagglutinating activity