3D illustration of a protein’s three-dimensional structure, with its components highlighted in different colors against a gradient background ranging from blue to black.

Application Note

Mastering membrane protein production

Explore a streamlined approach to protein expression and purification that dramatically reduces timelines and experimental bottlenecks.

Membrane proteins are ubiquitous cellular components that are responsible for a diverse range of cellular and molecular activities, including drug metabolism, antibiotic resistance, and lipid shuttling. Producing membrane proteins for research purposes is plagued with challenges related to detergents that can damage protein structure and function.

Download this application note to learn about:

An innovative cell-free protein synthesis system that eliminate obstacles to rapid, reliable, and efficient membrane protein production
Achieving high yield, purity, and reproducibility for drug-relevant membrane proteins
Scaling up the expression of drug-relevant targets

Sponsored by

Top Image Credit:

istock.com/ Christoph Burgstedt

Shortened timelines to soluble, active proteins Conventional methods to optimize and obtain protein can take weeks to months especially if the expressed proteins are not soluble, not stable or not active, resulting in a repeat of the above workflow with new constructs. This leads to frustration, high cost and time loss. An automated protein expression and purification system that performs 192 expression and 30 purification screen in 24 hours. Scale-up protein expression the next day to get protein in-hand for downstream applications. www.nuclera.com 1 Transformation 2 3 5 Cell lysis 6 Verify soluble expression Protein purification Weeks 2 days Order new Cloning plasmids 7 Analysis of purification μg to mg of proteins 4 Protein expression Insoluble/low yield. New variants (up to 50 constructs or more). Add to Basket The protein expression conundrum: Lengthy project timelines and laborious workflow The solution: The eProtein DiscoveryTM System Protein-in-hand in 48 hours Introducing the eProtein Discovery™ System 2 The eProtein Discovery™ system Introducing a benchtop system for rapid protein expression and purification screening. Using Nuclera’s eProtein DiscoveryTM system, scientists can evaluate expression screens of 24 constructs with customizable cell-free blends in parallel. The construct that gives the highest yield can be selected for an off-cartridge protein purification scale up, delivering microgram quantities of purified protein in-hand in less than 48 hours. Nuclera’s technology integrates cell-free protein synthesis (CFPS) and digital microfluidics on eProtein Discovery Cartridges, allowing rapid progress on protein projects through a benchtop, automated, high throughput protein access system. One system, incremental gains: Much faster μg-to-mg 100% better success Greatly reduce overhead Much smaller footprint 000 sqft nuclera Not Nuclera (E.coli) nuclera Not Nuclera (Screening 0f 192 expression conditions) $30k+ $5.5k 70% 35% nuclera Not Nuclera Months 48h nuclera Not Nuclera The costs to obtain protein is vastly reduced using the eProtein Discovery system as less equipment and expertise are required. One system, remarkably varied applications: Unprecedented drug targets Difficult to express proteins AI/ML protein engineering De novo protein design Developability assessment Amino acid labelling Introducing the eProtein Discovery™ System 3 Protein Discovery Workflow Streamline your path to protein-in-hand with 3 simple steps: ▷ Step 1: Design and prepare DNA constructs ▷ Step 2: Automated protein expression and purification screen on cartridge ▷ Step 3: Scale up on the bench Design & Prep Step 1 Step 2 Step 3 Express Purify Scale Design, order and prepare linear DNA expression constructs Automated data-generating screens to determine your path to soluble, purifiable proteins Scale up proteins to µg and mg scale Explore multiple expression combinations in parallel, on one instrument Protein variations Dimension 1 Dimension 2 Dimension 3 Solubility tag options Expression conditions ▷ Domain deletions ▷ Truncations ▷ Site directed mutations ▷ Different Isoforms ▷ Homologs ▷ P17 ▷ FH8 ▷ ZZ ▷ SNUT ▷ CUSF ▷ TRX ▷ SUMO ▷ No solubility tag ▷ Redox environment for disulfide bond formation: PDI/GSSG, GSSG ▷ Chaperones: DnaK Mix, TrxB1 ▷ Metal Ions: ZnCl₂, MnCl₂, CaCl₂ ▷ Co-factor mix: combination of NAD, Acetyl-CoA, FAD, SAM, PLP ▷ 3C Protease for in situ solubility tag removal Introducing the eProtein Discovery™ System 4 Evidence 1: Consistent and reproducible readouts Reproducible cartridge screen results were obtained across different runs. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 3C protease + Buffer 3C protease + PDI/GSSG 3C protease + TRXB1 3C protease + TRXB Buffer + Buffer Buffer + PDI/GSSG Buffer + TRXB1 Buffer + Zn2+ Purified yield (µM) Cell-free blend User 1 User 2 User 3 User 4 User 5 User 6 User 7 User 8 User 9 User 10 User 11 Target protein BTK (392-659) was screened for expression and purified yield measured on the eProtein Discovery instrument by 11 independent users, using multiple equivalent instruments. Coefficient of variation across instrument runs are calculated to be <15%, consistent with batch-to-batch variations observed with cell-based expression and purification systems. Reproducible and repeatable scale-up yield 60 50 40 30 20 10 0 User 1 Yield (µg) User 2 MDM2 User 3 User 4 User 5 7 6 5 4 3 2 1 0 User 1 Yield (µg) User 2 PTPN11 User 3 User 4 User 5 Scale-up reproducibility across 5 different users working on the same protein. Each purified protein was determined to be >95% pure using a GelAnalyzer. Yield was determined using a fluorescence complementation assay. Coefficient of variation across scale-up repeats is 13% (PTPN11) and 11% (MDM2), consistent with batch-to-batch variations observed in cell-based expression and purification. Introducing the eProtein Discovery™ System 5 Evidence 2: Proven success with drug targets The eProtein Discovery system was used to guide scale up of drug target proteins and has produced many purified proteins with high yields. Target protein Purified Scale Up Yield (μg from 200μL cell-free blend) 0.00 20.00 40.00 60.00 80.00 100.00 120.00 VEGF(1-165) PARK7(2-189) TTR(21-147,T80A) CALM1(44-149) NLRP3(2-93,S5D,K23E,K24E) BRCA1(1280-1530) GATA1(190-340) YIPP GAPDH IFN-g (23-166) I13R1(128-226) BTK(211-659) MDM2(380-479) B-RAF(155-227) KRAS(2-165) NNMT NF2L2(334-605) HRAS IDH1(2-73) PTPN11(3-528) AMPC(K331A) TLL1(730-1003) CD19(21-277) CLOQ PRMT1(2-284) CAT TGFB1(279-390) TYK2(871-1187) IDO1(2-359) ADH1A MAPK1(2-360) MCE-VACCA(2-333) IKZF1(2-196,284-519) CalA(31-462) FLT3(583-953) SAPI(237-419) GNMT CAS12A(E174R/S542R) SMYD3(272-428) IALP ALT MLKL PD-1(1-288) SIRT1(231-496) SRC DHFR-TS(Ala-facemut) JAK2(849-1124) HIF1A(1-850) DHFR GUCY2C LYN c-Jun MGAT2(84-447) Illustration of a subset of pharma-relevant target proteins expressed using eProtein Discovery. An average yield of 35µg of protein obtained from 200µL of cell-free expression reagents (177µg per mL). Introducing the eProtein Discovery™ System 6 Evidence 3: High purity, assay-ready protein a) Purity of obtained scaled-up proteins: ▷ Average purity: 92.59% ▷ Median purity: 96.75% 100 – 90 – 80 – 70 – 60 – 1 2 3 4 5 Purity % b) Lane Description 1 Ladder 2 No-DNA Control 3 Cell-free expression 4 Flow through 5 Purified Protein 1 2 3 4 5 Scale-up purity data. Guided by cartridge generated results, DNA construct and cell-free blend combinations with the highest yield were selected for scale-up expression and purification. (a) Purity data collected from 62 independent purifications, purity determined by SDS-PAGE and purity quantified on a GelAnalyzer. (b) Example of purified BRCA1 (2-620) showing purity >95%. Introducing the eProtein Discovery™ System 6 Introducing the eProtein Discovery™ System 7 >1000 proteins generated on the system Cytokines and Growth Factors - IFN-γ - Erythropoietin - VEGF - IL-2 - HER2 Kinase/Phosphatase - SRC - PINK1 - KSR1 - JAK2 - PGK - BTK - B-RAF Antibodies - VHH Others: - TLL1 - PRMT1 - PARK7 - DHX9 - BRCA1 Transcription Factors - GATA1 - C-Jun - IKZF1 Metabolic proteins - SIRT1 - NNMT - MDM2 - IDO1 - IDH1 - ADH1A Regulators - SMYD3 - PD-1 RAS pathway proteins - KRAS - MAPK1 - CALM1 - PTPN11 Transporters - PTPN2 - TTR - MLKL - STING1 77% Human 10% Bacteria 5% Animal 4% Plant 2% Fungus 2% Virus & other A subset of trending drug targets successfully obtained on the eProtein Discovery System as going to press, March 2024. Summary A common significant problem for the protein science community are the long lead times and massive efforts to optimize protein constructs in order to generate enough protein for drug discovery and biomedical research. We offer a quick solution with the eProtein Discovery system which enables users to automate protein expression and purification on the benchtop. Screen through 24 different DNA constructs in parallel using customizable cell-free blends to gain knowledge on expressibility and purifiability. Scale-up the next day to get assay-ready protein in-hand! Nuclera Technical Customer Service: Phone/WhatsApp Business: +44 1223 942 761 Email: techsupport@nuclera.com Offices: Nuclera UK (HQ): One Vision Park, Station Road, Cambridge, CB24 9NP, UK Nuclera USA: 1000 Technology Park Drive, Suite B, Billerica MA 01821, USA www.nuclera.com Copyright © 2025 Nuclera Ltd. All trademarks are the property of Nuclera, Ltd. Visit nuclera.com/legal for more info. Version number: 12