Protein Expression and Thermal Shift Assay

URL: https://cloud.ginkgo.bio/protocols/cfps-strep-purification-thermal-shift Status: Ginkgo Certified Price: $159/sample Turnaround: up to 12 days Throughput: Up to 88 constructs per run (1 column reserved for controls)

Overview

Combines cell-free protein expression, Strep-tag magnetic bead purification, and a Protein Thermal Shift Assay using SYPRO Orange into a single end-to-end workflow. Starting from a DNA template plate, the protocol expresses protein in a CFPS reaction, purifies the tagged product via Strep-Tactin magnetic beads, and characterizes thermal unfolding of the purified protein by extrinsic fluorimetry.

SYPRO Orange is a hydrophobic-binding dye whose fluorescence increases sharply as a protein unfolds and exposes buried hydrophobic regions during a controlled temperature ramp. Tracking fluorescence vs. temperature reports Tonset (where unfolding begins) plus up to three melting transitions (TM1, TM2, TM3) corresponding to distinct domains. These are standard developability parameters used to compare candidates, flag stability liabilities, and rank molecules for downstream development. Best suited for screening and ranking variants by thermal stability directly from DNA, where consistent Tm values across many samples matter more than absolute biophysical precision.

Input

• DNA Input: Linear DNA sequence
• Tag Orientation: N-terminal or C-terminal fusion (validated with C-terminal tags; success is protein-dependent)
• Format: Up to 88 constructs per run, 1 column of wells for controls

Output

• Cell-Free Reaction: 100 uL E. coli-based CFPS reaction per sample with process controls
• Yield Quantification: Absolute protein yield in eluate via A280, converted to mg/mL
• Thermal Stability Measurement: SYPRO Orange thermal shift in triplicate on purified eluate, reporting Tonset and up to three melting transitions (TM1, TM2, TM3) in deg C, where applicable
• Reporting & Data: PDF report with yield data, thermogram plots, called TM values, reaction condition metadata, and QC status

Automated Workflow

Phase 1 - CFPS Reaction Setup

1. Stamp DNA into CFPS mix (Agilent Bravo 96)
2. Incubate (Cytomat / Inheco)

Phase 2 - Mag Bead Purification

1. Dispense reagents and load samples (Biotek / Agilent)
2. Bind sample to beads (Bravo Shaker)
3. Wash beads (Agilent Bravo 96)
4. Elute purified protein (Agilent Bravo 96)

Phase 3 - Assay Plate Preparation

1. Transfer 1 uL SYPRO dye (Echo 525)
2. Stamp purified protein (Agilent Bravo 96)

Phase 4 - Plate Sealing & Thermal Ramp

1. Seal plate (Agilent PlateLoc)
2. Execute melt-curve protocol (Bio-Rad CFX Opus thermal cycler)

Ordering

• Number of Proteins: configurable
• Number of Replicates: configurable
• File Upload: CSV, Excel, FASTA, TXT, PDF, ZIP
• Additional Details: free-text field for special requirements

Use Cases

• Ranking protein variants by thermal stability (Tm/Tonset) directly from DNA
• Developability screening and stability-liability flagging
• Comparing domain unfolding across candidate libraries