High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells.

Agnieszka Chytła,Aleksander Czogalla,Aleksander F. Sikorski,Agnieszka Biernatowska,Weronika Gajdzik-Nowak

doi:10.3390/membranes11090715

Abstract

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

Highlights

membrane palmitoylated proteins (MPPs) are ubiquitously expressed, membrane-associated, palmitoylated proteins found at the cytosolic leaflet of the plasma membrane (PM) [4]
PEI is a cationic polymer that is commonly used in transient gene expression (TGE) as a transfection reagent due to its known advantages [38,41]
This study aimed to find the optimal transfection parameters for the maximized overThis study aimed to find the optimal transfection parameters for the maximized overexpression yield of naturally palmitoylated recombinant MPP1 in HEK-293F cells

Summary

Introduction

Membrane-associated guanylate kinases (MAGUKs) are widely expressed proteins in all types of eukaryotic cells [1]. This well-conserved group of scaffolding proteins includes several subfamilies that are involved in multiple cellular processes, such as cell polarity, cell signalling, and cell adhesion [2,3]. One of these subfamilies is named the membrane palmitoylated proteins (MPPs). MPPs are ubiquitously expressed, membrane-associated, palmitoylated proteins found at the cytosolic leaflet of the plasma membrane (PM) [4]

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