TY  - JOUR
A1  - Dondapati, Srujan Kumar
A1  - Lübberding, Henning
A1  - Zemella, Anne
A1  - Thoring, Lena
A1  - Wüstenhagen, Doreen Anja
A1  - Kubick, Stefan
T1  - Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems
JF  - Frontiers in pharmacology
N2  - Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.
KW  - membrane proteins
KW  - Sf21 lysates
KW  - microsomes
KW  - cell-free protein synthesis
KW  - proteoliposomes
KW  - transporter
KW  - ion channel
KW  - pump
Y1  - 2019
U6  - https://doi.org/10.3389/fphar.2019.00917
SN  - 1663-9812
VL  - 10
PB  - Frontiers Research Foundation
CY  - Lausanne
ER  - 
TY  - JOUR
A1  - Dondapati, Srujan Kumar
A1  - Stech, Marlitt
A1  - Zemella, Anne
A1  - Kubick, Stefan
T1  - Cell-free protein synthesis
BT  - a promising option for future drug development
JF  - BioDrugs
N2  - Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.
Y1  - 2020
U6  - https://doi.org/10.1007/s40259-020-00417-y
SN  - 1173-8804
SN  - 1179-190X
VL  - 34
IS  - 3
SP  - 327
EP  - 348
PB  - Springer
CY  - Northcote
ER  -