Plastids like chloroplasts contain their own circular DNA and are capable of expressing foreign genes at high levels. This document describes research where the human serum albumin (HSA) gene was successfully transformed into the tobacco chloroplast genome. Up to 11.1% of total soluble protein consisted of HSA, over 500 times higher than when introduced into the nuclear genome. The chloroplast transformation resulted in HSA accumulating in inclusion bodies, facilitating its purification. This demonstrated chloroplasts as an effective system for the high-level production of recombinant human therapeutic proteins.

1. By: Kanchan Rawat

3.  Plastids are cellular organelles with circular double
stranded DNA.
 Various forms of plastids are Amyloplast for storing
starch, Elaioplast for fat storage, Chromoplast for
pigment synthesis and storage and Choloroplast for
photosynthesis.
 Chloroplast origin is prokaryotic.

4.  ~900 chloroplasts per
plant cell
 Each cell contain ~10,000
identical copies of each
plastid gene.
 CpDNA is packed into
discrete structures called
chloroplast nucleoids.
 Genome size :
 120-160 kb
 Each plastid cell contain
120 genes.

5.  Risk of transgene escape: chloroplast genome is maternally
inherited so it provides gene containment thus reduces the
escape of transgene.
 Expression level: Higher level and multiple transgene
expression due to polycistronic mRNA.
 Homologous recombination: It minimizes the insertion of
unnecessary DNA that accompanies in nuclear genome
transformation.
 Gene silencing is absent.
 Disulphide bond formation and folding human proteins
results in high level production of proteins.

6. Nuclear genome Chloroplast genome
Gene silencing results in
decrease or elimination of
transgene expression.
Gene silencing is absent.
Paternal transgene inheritance
results in outcrossing among
crops and weeds.
Maternal gene inheritance in
most crop plants results in
natural gene containment.
Highly variable gene expression. Uniform gene expression.
Each transgene is independently
inserted and transcribed into a
monocistronic mRNA.
Genes transcribed into
polycistronic RNA so that
multiple transgenes can be
introduced and expressed in a
single transformation event.

7.  2 successful methods include biolistics and
polyethylene glycol mediated transfer.
 Biolistic DNA delivery is used when the targets in
plastids are intact tissue.
 Polyethylene glycol treatment is used for DNA
introduction into protoplasts.
 Biolistics is preferred as it is less time-consuming and
demanding.

9. The plastid genome segments that are included in the vector are
marked as the left (LTR) and right targeting regions (RTR). A
selectable marker gene and gene of interest is inserted in vector.

12.  HSA is synthesized in the liver and functions as a
carrier protein for many exogenous and endogenous
metabolites and drugs.
 It accounts for 60% of the total protein in blood
serum.
 It is the most widely used intravenous protein in a
number of human therapies.
 It is highly susceptible to proteolytic degradation in
recombinant systems and is expensive to purify.
 Very low expression levels of HSA were attained
(0.02% tsp) via nuclear transformation.

13.  The annual world need of HSA exceeds 500 tons.
 Only source of HSA is blood so there is chance of
transmitting pathogenic viruses.
 In addition, good recombinant systems are still not
available for many human proteins that are expensive
to purify or highly susceptible to proteolytic
degradation.

14. Integration of transgene cassettes into the chloroplast genome.
HSA is driven in all cassettes by the Prrn promoter upstream of the
aadA gene for spectinomycin resistance with additional promoters
and control elements.

15. Southern blot analysis.
b)Probe P1 and P2 used for southern blotting.
c)Lane 1:untransformed DNA; 2,3 :DNA from plants transformed with
pLDAsdHSA; 4,5: DNA from plants transformed with pLDApsbAHSA.
d)Plants for the first (T0) and second (T1) generation were analysed.
2,4: T0 generation. 3,5: T1 generation.

16. Analysis of HSA accumulation in transgenic chloroplasts.
(a) ELISA of HSA accumulation in leaves at different stages of development.
(b) Study after different hours of illumination. Samples of leaves were collected from
potted plants transformed with pLDApsbAHSA after the 8-h dark period or at
indicated hours in the light.

17. Colorimetric immunoblot detection of tobacco protein extracts
from mature leaves.
Total protein extracts were loaded in the gel. 1)pure HSA; 2: mw
marker; 3,5: untransformed plant extract; 4: pLDAsdHSA plant
extract; 6: pLDApsbAHSA plant extract.

18. HSA accumulation into inclusion bodies.
(a–d) EM of immunogold labelled tissues from untransformed (a)
and transformed mature leaves with the chloroplast vector
pLDApsbAHSA (b–d)

19. HSA extraction from inclusion bodies.
(a) SDS-PAGE gel showing 1: pure HSA; 2: marker; 3,4:soluble
fraction obtained after centrifugation of pLDApsbAHSA transformed
plant extract, 5: HSA after solubilization from the pellet; 6: proteins
from untransformed plant.

20. Immunoblot detection of protein extracts.
1: pure HSA; 2: HSA from a plant transformed with pLDApsbAHSA
during the solubilization process, showing mono, di and trimeric
forms; 3: proteins from an untransformed; 4: same HSA from lane 2
but in a more advanced stage of solubilization; 5: completely
monomerized HSA after the end of the solubilization treatment.

21. Plant T1 phenotypes
1,2: untransformed plants; 3: plant transformed with pLDAsdHSA;
4:plant transformed with pLDApsbAHSA.

22.  500 folds higher concentration of HSA was observed
than usual concentration.
 11.1% of tsp of HSA was observed as compared to
0.02% observed in nuclear transformation.
 Inclusion bodies facilitated purification of HSA .
 Regulatory elements eg. psbA 5’UTR served as a
model system for enhancing expression of foreign
proteins.

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