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

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.

LIX1-mediated changes in mitochondrial metabolism control the fate of digestive mesenchyme-derived cells

“…To determine whether LIX1 is S-palmitoylated, we transfected HeLa cells with wild-type HA-LIX1 (LIX1 WT) or LIX1 mutants in which cysteine 83 and 84 were replaced by serine residues (LIX1 C83S, LIX1 C84S). We then tested S-palmitoylation in total protein extracts using the acyl-RAC assay [ 30 , 31 ]. We detected a specific signal corresponding to S-palmitoylation in the cleaved bound fraction (cBF) of LIX1 WT and LIX1 C83S samples, but not in the LIX1 C84S sample ( Fig.…”

LIX1 controls digestive mesenchyme-derived cell fate decision by regulating cristae organization in mitochondria

“…To determine whether LIX1 is S-palmitoylated, we transfected HeLa cells with wild-type HA-LIX1 (LIX1 WT) or LIX1 mutants in which cysteine 83 and 84 were replaced by serine residues (LIX1 C83S, LIX1 C84S). We then tested S-palmitoylation in total protein extracts using the acyl-RAC assay [27,28]. A specific signal corresponding to S-palmitoylation was detected in the cleaved bound fraction (cBF) of LIX1 WT and LIX1 C83S samples, but not in the LIX1 C84S sample (Fig.…”

“…LIX1 S-palmitoylation was assessed using the acyl-resin assisted capture (Acyl-RAC) method and the CAPTUREome™ S-Palmitoylated Protein Kit (Badrilla, K010-310; Leeds, UK), as previously described [28]. Briefly, free thiol groups were first blocked.…”

Polyethyleneimine-Based Lipopolyplexes as Carriers in Anticancer Gene Therapies

“…Excess of PEI not only seems to prevent aggregation of such cationic polyplexes but might also improve transfection efficiency [ 70 , 71 ]. However, the optimal N/P ratio for efficient transfection is unique to each formulation, as it depends on various factors, such as size and topology of both of the polymers, ionic strength of the environment, and even the cell line to be transfected, some of which are discussed below [ 19 , 72 ].…”

“…Despite a plethora of studies focused on modification of such polycations to ensure their biodegradability, higher specificity, or stability, polyplexes still face similar difficulties as lipoplexes, with low transfection levels being the primary obstacle to therapeutic use [ 18 ]. It is worth underlining that, even in in vitro conditions, prolonged exposure of cells to PEI/DNA complexes may significantly impair cellular proliferation and viability [ 19 ].…”