A procedure for parallel purification of four stress-related Neurospora proteins in their native state.

Abstract

A procedure is described for isolation and purification of four high-molecular mass stress-responsive proteins from the same starting material. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol56/iss1/4 8 Fungal Genetics Reports A procedure for parallel purification of four stress-related Neurospora proteins in their native state. M. Kapoor. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada Fungal Genetics Reports 65:8-11 A procedure is described for isolation and purification of four high-molecular mass stress-responsive proteins from the same starting material. Proteins are the major components determining cellular growth, metabolism, regulation, repair, homeostatic mechanisms, differentiation, organogenesis, response to environmental factors and ultimately survival. Extensive protein networks form the basis of interlocking metabolic pathways and their regulatory circuits in the eukaryotic and prokaryotic cells, involving transient interactions among a large, overwhelming array of actors. Availability of purified proteins in their native state is critical on account of the universal interest in unraveling how proteins function in their endogenous intracellular environment. With the rapid progress in genomics and availability of an ever increasing number of crystal structures, an understanding of the molecular basis of interactions of proteins with ligands, substrates and regulatory molecules is achievable. Structural studies, for instance X-ray crystallographic analysis, depend on homogeneous preparations of proteins. The use of heterologous expression systems with bacteria as host cells is a well established methodology. Other host systems include yeast, insect cells and plant cells. The technology for cloning and expression of proteins, vectors with selectable markers, enhancers, trafficking signals and a range of facile protocols for over-expression are also readily available. However, often it is feasible to express only individual domains of large multi-domain proteins efficiently in heterologous systems. Even when the entire polypeptide can be expressed it is not always possible for the host cell to perform the necessary post-translational modifications. Furthermore, it is often difficult to obtain sufficient material— tissues/cells—to enable recovery of requisite quantities of the critical protein for physico-chemical analyses. If the source organism is easy to cultivate and adequate quantities of starting material can be acquired in a relatively short time, isolation and purification of the target protein(s) in the native state presents the best opportunity for insightful structural studies. On account of its rapid growth and simple nutrient requirements Neurospora is the ideal organism for acquisition of purified proteins. The following is a description of a procedure developed in my laboratory for isolation of highmolecular-mass stress-related Neurospora proteins (Fig. 1): the molecular chaperones nHsp70, nHsp80, heat-induced peroxidase (HI-per) and BrnE (Band running next to Hsp Eighty) subsequently identified as the oxidative stress-responsive, cobalamin-independent methionine synthase (MetS). This multiplex protocol results in the recovery of four proteins from the same starting material: common initial steps are followed by fractionation schemes specific to individual proteins, thereby generating near-homogeneous preparations. The general methodology entails standard protein precipitation, ion-exchange, hydrophobicand affinitychromatography steps (1). The overall yield of the final products is in milligram quantities, sufficient for identification by MALDI-TOF MS analysis, examination of physico-chemical properties and structural studies. Details of the protocol are outlined in the accompanying flow chart. Figure 1. Autoradiographs depicting SDS-PAGE profile of non-shocked (A) and heat-shocked mycelial cells (B), grown for 14 h and labeled with [S] methionine for 1 h during heat-shock treatment. 1 Kapoor: A procedure for parallel purification of four stress-related Neur Published by New Prairie Press, 2017