Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Summary

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Abstract

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Introduction

Since the 1970s, plants have been explored as alternatives to mammalian, insect, and bacterial cell cultures for the commercial production of recombinant proteins and protein therapeutics ¹. Plant-based systems for the expression of biopharmaceuticals have shown promise in recent years as several novel treatments for diseases, like Gaucher's Disease ², and avian H5N1 influenza ³, have shown success in clinical trials. The development of competent mechanisms for recombinant protein expression in plants in the decades since those initial experiments has created the potential for plant-based systems to alter the current paradigm of protei....

Protocol

1. Plant Growth

1. Place 60 peat pellets into a propagation tray. Add 4 L of tap water and let the peat pellets absorb the water for 2 hr.
2. Add 2 N. benthamiana seeds into each peat pellet using a seeder, cover the tray with a transparent plastic dome, and allow them to germinate in a 25 °C, 84% humidity environment with a 16/8 hr day/night cycle (Figure 1A).
3. Remove the dome two weeks after seeding, drain water from the tray, and add 2 L of Jack's fertilizer at a concentration of 1.48 g/L (Figure 1B). Continue to grow plants in a 25 °C, 50% humidity environment with a 16/8 hr day/night cycl....

Results

1. Expression of Fluorescent Proteins by Syringe Infiltration

To demonstrate the effectiveness of syringe infiltration of Agrobacterium into plant tissue, we tested the expression of two fluorescent proteins - GFP and DsRed - by two different deconstructed plant viral vectors - geminiviral and MagnICON - in N. benthamiana. For N. benthamiana leaves that were entirely infiltrated with Agrobacteria containing geminiviral vectors, GFP expression was observed over .......

Discussion

The increasing demands for protein-based pharmaceuticals worldwide require new production platforms that are robust, scalable, low-cost and safe. Plants have shown to be one of the most promising alternative production systems for pharmaceutical protein production. In recent years, the development of deconstructed virus-based vectors has enabled transient expression of proteins in plants, which greatly enhances the speed and yield of plant expression systems ^2,10. To further optimize the utility of the transie.......

Materials

Name	Company	Catalog Number	Comments
			Reagents
GFP	Invitrogen	V353-20	www.invitrogen.com See reference: Lico and Chen, et al 2008
DsRed	Clontech	632152	www.clontech.com See reference: Baird and Zacharias, et al 2000
MagnICON Vector	Icon Genetics	n/a	www.icongenetics.com See reference: giritch and Marillonnet, et al 2006
Geminiviral Vector	Author's Lab	n/a	See reference: Chen and He, et al 2011
N. benthamiana	Author's Lab	n/a	herbalistics.com.au
Agrobacterium tumefaciens strain gv3101	Author's Lab	n/a	See reference: Lai and Chen 2012
LB Agar Carbenicillin-100, plates	Sigma	L0418	www.sigmaaldrich.com
LB Agar Kanamycin-50, plates	Sigma	L0543	www.sigmaaldrich.com
Magnesium sulfate hepa hydrate	Sigma	M2773-500 g	www.sigmaaldrich.com
Bacto-Tryptone	Fisher	73049-73-7	www.fishersci.com
Bacto Yeast Extract	Becton, Dickinson & CO.	REF 212750	www.bd.com
Difco Nutrient Broth	Becton, Dickinson & CO.	REF 234000	www.bd.com
MES hydrate Buffer	Sigma	M8250-1kg	www.sigmaaldrich.com
Carbenicillin	Sigma	C1613-1ML	www.sigmaaldrich.com
Kanamycin	Sigma	70560-51-9	www.sigmaaldrich.com
Sodium Hydroxide	Sigma	221465	www.sigmaaldrich.com
Jack's Fertilizer	Hummert International	Jul-25	www.hummert.com
			Equipment
Vacuubrand MD4 Vacuum Pump	Fisher	13-878-113	www.fishersci.com
Vacuum Air Regulator Valve	Fisher	NC9386590	www.fishersci.com
Desiccator 12 1/8" with O ring	Fisher	08-594-15C	www.fishersci.com
3 L Tub	Rubber-Maid	n/a	Rubbermaid Servn' Saver Bowl, 10-cup will work
plate/shelf 230ML	Fisher	NC9489269	www.fishersci.com
Peat Pellet	Hummert International	14-2370-1	www.hummert.com
Propagation Tray Dome	hydrofarm	132052	www.hydroponics.net
Propagaiton Tray	hydrofarm	138758	www.hydroponics.net
Virbo Hand Seeder	Gro-Mor INC	n/a	www.gro-morent.com
Flora Cart 4 shelf	Hummert International	65-6924-1	www.hummert.com
15 ml Round Bottom Culture Tubes	Sigma	CLS430172-500EA	http://www.sigmaaldrich.com
Spectrophotometer	Bio-Rad	170-2525	www.bio-rad.com
Spectrophotometer Cuvettes	Bio-Rad	223-9950	www.bio-rad.com
Microcentrifuge Tubes	USA Scientific	1415-2500	www.usascientific.com
Benchtop Centrifuge	Bio-Rad	166-0602EDU	www.bio-rad.com
Incubator/Shaker	Eppendorf	Excella E25	www.eppendorf.com
Ultraviolet Light Model#: UVGL-25	UVP	95-0021-12	www.uvp.com