Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing

Abstract

G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

Production of G protein‐coupled receptors in an insect‐based cell‐free system

“…S1 and Table S1). Although a CECF reaction is approximately 10 times more expensive compared to a typical batch reaction an increase of the total protein yield up to 100‐fold was observed (Merk et al, ; Quast et al, ). Therefore, it had proved possible to achieve a significant reduction of the total costs by a factor of 10 within the last years.…”

“…A major disadvantage of these systems is the required addition of a suitable detergent to solubilize and stabilize de novo synthesized membrane proteins (Bernhard and Tozawa, ). Furthermore, many GPCRs require posttranslational modifications (PTMs) such as phosphorylation, palmitoylation, glycosylation, and disulfide bond formation to stabilize their active state and correct folding (Klammt et al, ; Merk et al, ). Neither E. coli nor WGE contain the necessary machinery to ensure complete posttranslational protein processing.…”

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…146 The development of cell-free systems depends upon both qualitative and quantitative considerations. Qualitative control may be achieved through use of insect cells, 153,154 or HeLa cells, 155 to overcome improper protein folding or glycosylation. The quantitative aspect of these systems, however, remains the production bottleneck.…”

Unexpected instabilities explain batch‐to‐batch variability in cell‐free protein expression systems

“…Several other sources for cell-free systems have also been studied, such as those from insect cells (Kubick, Gerrits, Merk, Stiege, & Erdmann, 2009;Merk et al, 2015;Stech et al, 2012), yeast (Gan & Jewett, 2014;Hodgman & Jewett, 2013;Schoborg, Hodgman, Anderson, & Jewett, 2014) …”

“…Several other sources for cell-free systems have also been studied, such as those from insect cells (Kubick, Gerrits, Merk, Stiege, & Erdmann, 2009;Merk et al, 2015;Stech et al, 2012), yeast (Gan & Jewett, 2014;Hodgman & Jewett, 2013;Schoborg, Hodgman, Anderson, & Jewett, 2014) and the protozoan lizard parasite Leishmania tarentolae (Gagoski et al, 2016;Johnston & Alexandrov, 2014;Kovtun, Mureev, Johnston, & Alexandrov, 2010;Kovtun et al, 2011;Mureev, Kovtun, Nguyen, & Alexandrov, 2009;Ruehrer & Michel, 2013), each with their own advantages and disadvantages. We routinely use the L. tarentolae system, since it offers the low cost, ease of culture and simple cell-free lysate production of the E. coli system, with the ability to more faithfully express eukaryotic proteins (Gagoski et al, 2016).…”