Home › Analytical Chem › High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Analytical Chem JoVE (Open Access) Citable · DOI

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

DOI: 10.3791/51464-v
What you'll learn
  • ✓ Design and execute high-throughput expression screening of recombinant proteins in E. coli cultures
  • ✓ Apply analytical purification techniques to disulfide-rich venom protein targets
  • ✓ Quantify protein expression levels across multiple construct variants systematically
Protocol

Biopharma Insights A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Difficulty
advanced
Total time
~5–7 days (expression screening 2–3 days, purification and analysis 2–3 days, depending on construct library size)
Biosafety
BSL-1

Steps

1
Perform test expression of recombinant venom proteins

Set up small-scale E. coli cultures for multiple constructs and induce protein expression. Monitor expression levels to assess solubility and yield across the variant library.

▶ 01:27
2
Purify and analyze expressed fusion proteins

Apply analytical purification methods to isolate recombinant disulfide-rich proteins from crude lysates. Characterize purity and yield using appropriate separation and detection techniques.

▶ 05:32
3
Interpret high-throughput expression screening results

Evaluate expression data across the 96-protein venom library to identify successful constructs and expression conditions. Use quantitative metrics to rank candidate proteins for downstream applications.

▶ 09:22
💬 Comments coming soon