Home › Immunology › Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Immunology JoVE (Open Access) Citable · DOI

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

DOI: 10.3791/51204-v
What you'll learn
  • ✓ Optimize Agrobacterium-mediated transient protein production in Nicotiana plants
  • ✓ Execute vacuum infiltration protocol for efficient protein expression
  • ✓ Analyze and quantify target protein accumulation using Western blot and imaging
Protocol

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Difficulty
intermediate
Total time
~7–10 days (plant growth 5–7 days, infiltration and harvest 1–3 days)
Model organism
Nicotiana species (tobacco)
Biosafety
BSL-1

Steps

1
Cultivate and prepare Nicotiana host plants

Grow Nicotiana plants under controlled conditions to appropriate developmental stage for infiltration. Select optimal host species to maximize target protein accumulation.

▶ 02:48
2
Perform vacuum infiltration with Agrobacterium suspension

Prepare Agrobacterium cultures carrying TMV-based launch vectors and co-introduce RNA silencing suppressors. Apply vacuum infiltration to submerge plants and facilitate bacterial uptake into leaf tissue.

▶ 03:55
3
Conduct Western blot analysis for protein detection

Extract plant tissue protein, separate by gel electrophoresis, and detect target protein expression using antibodies to quantify accumulation levels.

▶ 07:41
4
Image and visualize fluorescent protein expression

Use GFP imaging to visualize spatial distribution and intensity of expressed fluorescent proteins within infiltrated leaves.

▶ 09:40
5
Evaluate infiltration efficiency and protein yield

Compare target accumulation across Nicotiana species and growth conditions. Validate that optimized Agrobacterium infiltration achieves rapid, economical vaccine antigen and therapeutic protein production.

▶ 10:21
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