Molecular Farming: Producing Human Somatotropin in Transplastomic Tobacco Plants

Introduction to Molecular Farming and Human Somatotropin

Molecular farming harnesses plants as biofactories for producing therapeutic proteins. This lecture focuses on producing human somatotropin (HST), a pituitary-derived hormone essential for growth, within tobacco plants using chloroplast genetic engineering.

Why Human Somatotropin and Transplastomic Plants?

HST Significance: Naturally produced in the pituitary gland; requires precise post-translational modifications (e.g., disulfide bonds) to be biologically active.
Transplastomic Approach: Integration of HST gene into the plastid genome of plants enables high-level protein expression and maternal inheritance, reducing gene flow risks.

Gene Modification Strategy

Challenge: Plant translation typically initiates with methionine as the first amino acid, whereas human HST starts with phenylalanine post signal-peptide removal.
Solution: Fusion of HST gene with a ubiquitin (Utin) gene to alter the N-terminal amino acid sequence and mimic animal-type protein. This strategy is akin to methods explored in Metabolic Engineering of Indole Alkaloid Biosynthesis: Case Studies in Plants and Yeast where gene fusion techniques enhance biosynthesis pathways.

Promoter Selection for Enhanced Expression

Strong Promoters Used:
- PpsbA promoter (moderate strong promoter)
- PrrnG10L promoter (ribosomal RNA operon promoter with G10 leader sequence) with stronger expression capability

Optimizing promoters parallels advances in Metabolic Engineering Enhances Alkaloid Production in Catharanthus Roseus Hairy Roots, where promoter selection critically impacted expression levels.

Construct Design and Plant Transformation

Three constructs were developed:
1. Native HST under PpsbA promoter
2. Utin-HST fusion under PpsbA promoter
3. Utin-HST fusion under PrrnG10L promoter
Transplastomic tobacco plants were successfully generated and confirmed via Southern blot analysis.

Protein Expression and Verification

Western Blotting: Confirmed HST accumulation with expected protein sizes; fusion proteins exhibited two distinct bands.
Post-translational Processing Analysis: Disulfide bond formation validated through western blot under reducing and non-reducing conditions.
Mass Spectrometry: Confirmed molecular identity of the expressed proteins.

Biological Activity Test

Cell Proliferation Assay: Purified HST induced significant proliferation in NB2 cell lines, with fusion proteins showing enhanced activity over unmodified HST.

Expression Levels Achieved

Nuclear gene integration with chloroplast targeting yielded 0.025% of total soluble protein.
Unmodified HST in chloroplasts raised it to 0.2%.
Utin-HST fusion with PpsbA promoter reached 1%.
Utin-HST fusion with PrrnG10L promoter achieved up to 7%, representing a 35-fold increase over nuclear systems.

Biological Containment and Maternal Inheritance

Reciprocal crossing experiments confirmed that transgenes are maternally inherited, preventing transgene escape via pollen.
Progeny seeds lacking plastid inheritance showed sensitivity to spectinomycin, indicating effective containment.

The biological containment strategy aligns with principles discussed in Metabolic Reprogramming in Catharanthus Roseus for Non-Natural Indole Alkaloids, emphasizing secure transgene containment in plant biotechnologies.

Conclusion

Successful modification of the HST gene and promoter optimization led to high-yield, biologically active human somatotropin production in tobacco plastids.
The maternal inheritance characteristic ensures biosafety through biological containment.
This study exemplifies the potential of transplastomic plants for producing human therapeutic proteins in a cost-effective, scalable manner.

Reference

De Cosa, B., Moar, W., Lee, S. B. et al. (2000). Overexpression of the human somatotropin gene in transplastomic tobacco plants. Nature Biotechnology, 18(3), 333–338.

[Music] [Music] welcome to nptl online certification

course on pharmacognosy and metabolic engineering this is the last lecture of this course which is lecture number 63

so this is molecular farming so where I will discuss the production of human somatotropin hormone in Plants

particularly in tobacco so the concept covered is production of human somatotropin in transplastomic plants so

as you uh understand from this slide that uh so the approach is basically tar the the gene to be targeted into the

plastic so somatotropin why somatotropin so there are few questions

Som sorry Som

tropin human somatotropin so the abbreviation is HST so somatotropin is basically produced in the pituitary

gland and uh what happens that this is basically the final form of hormone so

uh so the somatotropin which is produced in the pitutary gland will undergo

uh post transational processing which

includes this dulfi Bond formation and eventually the processed HST is

produced prss processed which is biologically

active now uh point is this that when this uh so okay uh one one uh

theoretical aspects let me tell in in pitutary gland the removal of signal peptide from

somatotropin which leaves Phile alanin as the inter terminal amino acid so in pituitary

gland removal of of signal peptide

from somatotropin it leaves phy

alanin as the first

interal amino acid now if

uh uh Norm but in the plant system what happens in the normal plant it is basically the

methionine whever in the plant translation process the first amino acid is

methionine that means the normal translation in

plastid basically that initiates at methionine as first inter terminal amino acid

so this to be taken care of if it should be of animal type so the methionin should not be the first inter terminous

amino acid therefore if the gene for human somatotropin is available say HST so that if the HST Gene is there so

this to be modified in such a way that it it may be it to be added with

a with another protein which is utin so so utin

HST fusion protein to be made where this will make

the first intermal amino acid uh no methin so rather I put

here okay but if only HST is used in the pl system so in normal course it will

make methon as the first interal amino acid so therefore the modification has been made in such a way that iuin gen is

fused so that it it it encodes other amino acids apart from methionin so that is important so that

modification they have made and other important thing what they have done they have used two different type of

promoters one is a strong promoter and another is a very strong

promoter the strong promoter what they have used that is p

psba and the other one is called uh uh

p r rn/ g1l so PRN Gil is basically a much

stronger promoter so basically it is ribosomal RNA operon promoter and G10 is the leader sequence if that promoter is

there so that is expected to provide much more stronger expression than the traditional

ppba okay so this is only the things I need to mention and then we'll go to the construct so there are three construct

showing here the first one is basically the without any modification that means normal human

somatotropin under the control of this ppsb promoter and this Terminator RPS 16 okay and the second one is basically

the fusion but under the normal promoter strong promoter ppsb but the third one is basically it's a much more stronger

promoter which is prrn Gil and the TPS so there are basically there will be three type of transplastomic plants one

is unmodified HST another one is modified HST with a moderate promoter and the other one is the modified HST

with a strong promoter and then uh this to be checked what is going to happen so the accordingly the this is the

vector where this is the multip triple cloning site here so Gene was inserted here as you see it is here and here

basically they are showing the different fragments uh which was required for southern analysis but we are not going

to discuss that so simply showing here that IU HST fusion protein is basically now in the vector and in addition you

have the a a because a is required for the selection okay so

the next one is this that uh uh okay they they successfully produce the transplastomic plants they have done

Southern analysis but we I am not showing that simply i showing the accumulation of HST protein so the

purified HST protein was used uh for making the antibodies so they have used this is a western blot and they're

showing the accumulation of HST protein here so there are uh different lines so first one this is basically the A and

this is basically the B so a is a nuclear trans NT 4747 and the plased HST this is 48 48

three so this things so this are basically from the pled and

uh so what you see the level of expression in the level of expression in

the pled is much stronger when you compare with this

one okay and the B is basically where they have used the stronger promoter PRN G10 l so that

basically showing uh more strong

longer HST so this is basically they have standardized the process and how they could get the uh more uh uh intense

signal process okay so that means through this Western blotting they have confirmed indeed the HST protein of

correct size is produced okay so because the size will be different when etin is attach and

when utin is not attached so that has been shown here with two bands okay next is that that let us considered the

transplastomic plants uh were successfully produced and they have confirmed the presence of this HST and

HST Fusion as the case may be because there are three three different sets one is normal HST one is HST fusion with a

moderate promoter and another is HST fusion with a strong PR promoter so in all the cases the transpost plant

produce the HST as appropriate and then what is important that once the HST produced it is important to see that

whether they are processed or not because as I have said that the uh somatotropin requires post transation

processing which includes DPI Bond formation so in order to check that what they have done is that they have uh they

run the hlc of the protein they have uh and where uh what they have seen that when uh the protein the they treated

with dtt plus or dtt minus so when uh you see that when it's treated with the dtt

plus uh so this is with the dtt plus and when is the dtt minus that is a difference so this difference indicates

this is dtt minus so this difference basically indicates that indeed the reducing agent diol

makes modification in the uh disulfide Bond formation if that disulfide Bond formation is not there DT treatment will

not show any changes at at it showing changes that indicates that the disulfate bond formation occurs so that

means the uh transplastomic plants the HST is basically processed within the plastid okay and next what they have

done is basically they they confirm this protein by this Mass

spectrometry uh and next what they have done that they uh another important thing they

have done is basically they check that if the if the uh plased

expressed HST is

biologically active that is their question so that means how to test that when you have the purified protein HST

so that HST they have uh used it in a Cell line called nb2 cell nb2 cell line so the nb2 cell

line was used so basically when this HST or UB HST

protein was added so this cell line shows uh proliferation so that means that

uh rapid proliferation because somatotropin basically stimulate cell division so

rapid proliferation of nb2 cell line was noticed when the ubhs or HST is used so obviously UB HST showed a better

response than the normal one so this indicates that the chlorop transplastomic plant produced HST is

indeed biologically active otherwise if it is not biologically active it will not show

iation okay so that part is fine so the next part is that that how much is the protein so that just to give you an

estimate that when uh when the gene was localized in uh nucleus and this is the

plasmid and uh so the gene localized in the nucleus and uh you put a signal peptide sequence so that the protein

moves into the chloroplast so there the the expression of this protein in terms of percentage of total soluble protein

is only 0.025% okay okay where where this protein nuclear transform protein was

targeted to endoplasm reticulum it is again much much less but when the normal

one that is the unmodified HST was targeted to chloroplast was was unmodified sorry

unmodified HST was Gene was transformed into uh Gene was used to make transplastomic plants so the

transplastomic plants in the chloroplast when they have checked uh the the content was

0.2% okay and when when HST Fusion was used using a moderate promoter this level raised up

to 1% and when strongest promoter was used this

level goes to 7% you see from 2 to 7% how much fold it has increased I think more than 35 fold or

so something like that okay so uh next point is that one important properties as I have mentioned that maternal

inheritance that means the transplastomic plants poen should not contain the gene HST Gene so that is the

advantage that when the poen are escape the hn3 gene will not Escape along with that so in order to check that they have

done reciprocal Crossing what they have done they have taken the seeds of wild type as well as the seeds from this

transplastomic plants like they have chosen 48 38 that is the best line so what they have done and they have made

Crossing first of all this is the female sign sign and this is the male sign so they have made

Crossing between this and you see here when wild type female is crossed with transplastomic male then what will

happen the uh the seeds when they started ger it germinates but why it is bleach because all this contains okay I

forgot to tell all this contains spectinomycin in the medium so these medium

contains spectinomycin or streptomycin okay so now you you have

the seeds you grow it in the MS medium containing spectrom isin so what will happen in this one what I am focusing on

this particular one so it may start germinate then eventually it will bleach why because there is no a a gene is

there so the spectrom will be absorbed by the plant and immediately it will bleach the tissue okay because although

you have the NT 4838 but that is a male one which was crossed so the

gene a a is not there in this so it is basically maternally inherited the PO of the PO of 4838 was used in crossing of

the wild type female as a result of that this DEC combinant is produced so where there is no Gene similarly in this one

this is both the while type so that there's no question of gen So So eventually it will die now let us see

the case here here while type is male however the the female contains the

transplastomic so that means the wild type poen was used and that will basically fertilize the NT 483 which is

transplastomic as a result of that recant is produced where the a gene retains in the female side so it

retained so when the crossing was made the recombinant it retained the a as a result of that it

survived containing it survived in the medium containing spectrom same is true for this one here both one is male and

one is female so both of them contain the transplastomic but the more point is that female have the transplastomic so

therefore this one is also survived so this is called biological Contex containment so these indicate that

indeed these are transplastomic plants and it is safe to grow the plants in the field so that it will not

cross-pollinate other plants in the nearby field and lead to the creation of super weed so that is why we call

chloroplast trans gen are maternally inherited so that property has been also tested so therefore

to uh end this one so what I said that human somatotropin gene they have modified in such a way so that it when

the first translation Amino upon upon translation the first amino acid will not be methine

but anything then apart from ethin will be produced and they made this by fusing HS utin before the HST and if HST is not

there it will make the methon so because we want to make more of the animal type because somatotopy not the plant type so

therefore this Fusion was required and it worked well so the gene was successfully inserted they have checked

the size they check they later check the expression and they also check the HST fusion protein formation by Western

blotting and then the protein was tested for its uh post transational processing Properties or Not by dtt treatment then

protein was was also tested for its cell proliferation capacity so that also successfully passed and finally they

have tested the biological containment whether these are indeed the transplastomic proteins or not so that

is that know the plants are indeed transplastomic and the protein is produced this and we have also shown the

level of expression uh it's much much higher than that of the nuclear transformation and why that I have

explained in the previous class so this is basically a very successful example of high yield production of human

therapeutic protein in in tobacco and this paper published in uh nature biotechnology I'll just give you the

reference although this is an old paper but this is the best paper to tell the positive results of

transplastomic so this was published in the year of 2000 uh volume 18 in the March issue page

number 333 to 3 uh

38 so if you are interested you can get this paper simply this is available in the research gate so if and the first

author is 12ab is T Au U St all and and this was basically basically

uh this work was done at Monsanto company and agus Company this is not from academic Institute but a private

laboratory so Monsanto is the Giant in in in agricultural biotechnology so they ventured into the field and uh they

produce this work so and that this is a very successful case of transplastomic plants and particularly for the teaching

in the class I always prefer prer to discuss this paper rather than other papers so that students can really

understand the beauty of this transformation so with this I end this class and I end this course hope you

have uh enjoyed this course although a bit heavy but I try to cover a lot of things but of course uh there are few

things which remained left out but maybe in future when I'll modify this course I will add it up

so hope you'll be able to make uh to submit the assignments and I expect that a sizable number of the students will

write the exam and they will collect the certificate which may be useful if they are interested to go for higher studies

so such certificate will be useful at least because this is the full course and you'll be able to answer questions

if asked during your uh PhD or integrated PhD interviews or wherever may be postdoctoral anything

cases so knowledge is always be wealth for you with this I end this class thank you very much for your uh patient uh

learning going through the process thank you

Heads up!

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