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Lecture 2. Transgenic Plants Viral Vectors Transient Expression. Expression in Transgenic Plants. Limitations some crops can not be regenerated vulnerable to weather not 100% biologically contained unless via chloroplast transformation. Advantages low initial investment

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lecture 2
Lecture 2
  • Transgenic Plants
  • Viral Vectors
  • Transient Expression
expression in transgenic plants
Expression in Transgenic Plants

Limitations

  • some crops can not be regenerated
  • vulnerable to weather
  • not 100% biologically contained unless via chloroplast transformation

Advantages

  • low initial investment
  • medium time scale (months)
  • unlimited scale-up potential
  • faster than transgenic animals
  • very low costs <5$/gram
  • safe, no risk of pathogen contamination
  • edible crops can be used
  • active complex proteins (sIgA)

1

viral vectors
Viral Vectors

Viral Vectors for the expression of proteins in plants

Yuri Gleba, V. Klimyuk and S. Mariollnnet

Current Opinioins in Biotech 2007 18: 134-141

viral vectors1
Viral Vectors
  • Plant viral vectors
  • Clone GOI into virus
  • Infect plant
  • Virus replicates
  • Protein of interest will be produced to high levels
viral vectors advantages
Viral vectors - Advantages
  • Quick (2-3 weeks)
  • High Yield of protein
  • Gene is not incorporated
      • Not heritable trait
      • Containment of genes
first generation viral vectors
First generation Viral vectors
  • Functionally Viruses
  • Express all viral genes and GOI
  • GOI can be fused to CP
  • GOI expressed from strong promoter
  • To get GOI into plants
      • Use DNA or
      • Viral particles
  • Rub or spray onto plants
  • Virus then moves systemically and whole plants gets infected and produces POI
slide13

Tobacco mosaic virus vector

Large Scale Biology Corp.

pBSG1057

disadvantages
Disadvantages
  • Only small epitopes are expressed
  • Low yield (0.04 – 0.3% TSP)
  • Instability of gene
      • Loss of trans gene after while
      • Depends on size of gene – bigger, loss quicker
  • Limited systemic infection
  • To increase expression put in linker between CP and GOI
second generation of viral vectors
Second Generation of Viral Vectors
  • Use live Agrobacterium as vector
      • Agrospray or Magnifection
      • Need to contain recombinant Agro
  • Make viral genome smaller
      • Delete all non-essential genes
        • Ability to infect host
        • Systemic infection
  • Replication generally good – keep
  • Changed viral vectors
      • Codon usage
      • Add plant introns
slide16

First Generation Viral Vectors

Second Generation Viral Vectors

viral vectors2
Viral Vectors
  • Add T-DNA
  • Use Agro to deliver proviral replicon
  • Active replicons in plant cell
  • Get amplification in each cell and
  • Movement from cell to cell
  • Got increase in yield:
      • g/kg tissue
      • 50% TSP
  • Quick 4-10 days
slide18

First Generation Viral Vectors

Second Generation Viral Vectors

slide19

Infiltration

Vacuum – 0.8 bar for 20 sec

viral vector gfp expression

6 Days Post-Inoculation

2 Days Post-Inoculation

4 Days Post-Inoculation

UV LIGHT

NORMAL LIGHT

VIRAL VECTOR GFP EXPRESSION
field production of vaccines

Inoculate on plant leaves

Harvest in 10 - 14 days

Empty Viral Vector

Empty Viral Vector

Laboratory Work: Few Days

Vaccine Gene

Vaccine Gene

Viral vector with vaccine gene

Extract & Purify

Time from Gene to Field Production: 6 Weeks to a Few Months

Field Production of Vaccines
expression with viral vectors
Expression with viral vectors
  • This method used for any proteins to date:
  • Human growth hormone – 1mg/kg
  • Plague antigen
  • TB antigen
  • Ab – could not express more than one protein form same viral replicon
  • Use two viral vectors (heavy and light chain)
slide23
TMV
  • ss RNA, 2100 CP
  • Get very high yield of CP in plants – 10g/kg
  • Fuse GOI to CP
      • Can only fuse peptides (20AA)
      • Linker – bit bigger
  • Fuse antibody-binding domain of Protein A to CP
  • Use to purify
slide24

TMV

Protein A fragments

Enzymes

Ab

the icon deconstructed virus system is the newest of a series of technology advancements
The ICON deconstructed virus system is the newest of a series of technology advancements

B

GFP

A

DSRED

slide26

Expression Technology Development over time

Gene encoding the desired protein.

Incorporate into a plant transformation vector for optimized expression in plant cells.

Stable expression: Nuclear genome integration.

Stable expression: Chloroplast genome integration.

Integrate into a viral coding sequence for expression as a “by product” of viral replication.

Transient expression: Infect plant to initiate viral replication.

Modify viral genome to adapt it into a plant transformation vector for subsequent regulated release as a replicon in transgenic plants.

Primarily for antigens/epitopes until very recently

Identify protective epitope within antigenic protein.

Create viral replicon coding sequence with epitope fusions to coat protein.

transient expression
Transient Expression
  • Use Agrobacterium infiltration
  • Plant expression vectors
  • Advantages
      • can express more than one gene in same cell
      • Quick – 3-10 days
      • High yield
      • Can produce transgenic plants with same constructs
      • T-DNA can integrate or remain as episome
ptra plant expression vector

P35SS

5’UTR

GOI

(SEKDEL)

pA35S

SP

LB

pAnos

ColE1 ori

Pnos

selectable marker

bla

SAR

pTRA

RK2 ori

P35SS

gene of interest

RB

5'UT

SAR

pA35S

pTRA Plant Expression Vector
  • P35SS: CaMV35S promoter withdoubled enhancer
  • 5’UTR: tobacco etch virus 5’UTR
  • SP: signal peptide usedfor mammalian expression ofthe antibody chains
  • SEKDEL: C-terminal ER-retention signal (optional)
  • pA35S: CaMV35S terminator
  • SAR: scaffold attachment region
slide30

Agrobacterium-infiltration as a useful tool for rapid assessment of gene expression in plants

LB

RB

35S

Gene of Interest

OCS

[kDa]

97

69

46

wt 945(P) A 945 (A)

1 2 2d 5d

HPV L1

Western blot

analysis

slide31

Plant Vaccine Group University of Cape Town

direct injection

vacuum infiltration

Optimising protein production in plants

1. Select optimal constructs using agroinfiltration - simple and rapid method for evaluating protein expression of numerous Agrobacterium constructs

2. Small OR Large-scale infiltration