Production of Biologically Active Human GM-CSF Protein in Rice Seeds

Katie Surckla

hGM-CSF stands for human Granulocyte-Macrophage Colony Stimulating Factor

hGM-CSF is a cytokine which regulates the production and function of white blood cells.

It is found in the body, but in very low concentrations

This protein is helpful for fighting a variety of infections.

What is Human GM-CSF?

Neutropenia

Pneumonia

Crohn’s fistulas

Diabetic foot infections

HIV-related opportunistic infections

Given to bone marrow transplant patients

hGM-CSF clinical uses

Majority of these proteins are produced in mammalian cells, or single celled organisms like yeast and bacteria and insects.

However, these methods are extremely expensive.

Ex.-Mammalian cell-based manufacturing facility can cost up to $250 million!

Therapuetic Recombinant Proteins

What else could we use these for? Breast milk proteins hGM-CSF is one of the many proteins that

are looking to be added to breast milk by oral ingestion of recombinant proteins by the mother

This research is being done for mothers with HIV to resist passing on HIV to their children

Recombinant proteins

These methods can be challenging and hard to obtain high levels of expression of this protein

Public perceptions of these challenges

Getting approval to use land to field grow these transgene plants

Possible of inadvertent contamination of the food supply

Under stringent quality control and safety standards

Major barriers

Seeds are the most appealing target tissue

Seeds naturally store stable proteins for long periods of time

A large proportion of seed proteins belong to small sets of protein classes which helps in the purification steps.

Rice is also a popular weaning food for infants

Why rice seeds?

Rice endosperm cells

Typical Rice Endosperm

A 1.8 kb rice endosperm-specific glutelin promoter (Gt1) was used

Standard DNA cloning and DNA amplification techniques followed.

Plasmid contains:

Gt1 promoter

72 bp Gt1 signal sequence

*Transgene sugarcane production used particle gun bombardment while transgene rice seeds were produced in culture with a binary vector

DNA cloning

1) Digestion with NaeI enzyme

2) Dephosphorylation

3) hGM-CSF coding DNA sequence in BBG12 plasmid was amplified

4) DNA sequence was phosphorylated

DNA cloning steps

5) The plasmid was involved in a ligation reaction with the Gt1 promoter, glutelin signal sequence and the GM-CSF DNA fragment.

6) The transformed colony was identified

7) The plasmid was cleaved with BamH1 and HincII enzymes

DNA cloning steps

8) This plasmid contained an EcoR1 site on the 5’ end and a NOS terminator sequence at the 3’ end.

9) A HindIII site was added to the 5’ end of the Gt1 promoter

10) This fragment was cloned into a binary vector, pCAMBIA 1301.

DNA cloning steps

Agrobacterium strain LBA4404 was transformed with the binary vector pCAMBIA 1301

Callus induction of rice seeds, callus selection and plant regeneration were performed and the plants were allowed to grow to about 8 inches.

The plants were then grown in pots in a controlled chamber at 28° C and about 50-60% humidity.

Rice transformation, culture and growth

The rice genomic DNA was then isolated and purified

Southern blot-approximately 10 μg of rice DNA was digested and separated on 0.8% agarose gel, denatured then transferred to a nylon membrane.

The membrane was probed with 32P labeled fragment

Southern blot and PCR

Fig. 1-Southern Blot analysis on genomic DNA from rice plants. Lanes 1 and 2: positive control as HindIII insert released from the construct. Lanes 3-8: HindIII-cleaved genomic DNA from independent transgenic rice plants.

Clarified seed extracts were separated on 15% SDS polyacrylamide gels.

The proteins were transferred to PVDF membranes and treated with a blocking buffer solution.

Protein bands were visualized using the NBT/BCIP substrates.

Western blotting

Lanes 1-2: E. coli derived GM-CSF at 2 different concentrations.

Lanes 3-4: Non-transgenic plants Lane M: Prestained molecular weight marker Lanes 5-7: Transgenic rice plants of different

concentrations

Western blot results

The 1.8 kb Gt1 glutelin promoter from rice was used to control the expression of the hGM-CSF mature coding sequence.

The glutelin signal sequence was ligated in-frame with the coding sequence of GM-CSF.

Finally cloned into a binary vector, pCAMBIA 1301 which was then transferred to the competent LBA4404 strain of Agrobacterium

Gt1 promoter

The Agrobacterium cells were then used to transform vigorously growing rice calli.

6 transgenic plants regenerated from calli

PCR was used to verify the presence of the hGM-CSF sequence

Furthermore, the DNA was digested with HindIII to verify the integration into the rice genome

Integration of GM-CSF DNA in the rice genome

An hGM-CSF specific ELISA assay was used

1.2% tsp for plant #1 (28 μg/ml GM-CSF)

1.3% tsp for plant #2 (28 μg/ml GM-CSF)

Western blot also showed bands at 18 kDa (weight of GM-CSF in the non-glycosylated form)

Detection in rice seeds

Was tested using a human cell line, TF-1

This medium only grows in the presence of hGM-CSF or other growth factors

Assay medium alone (without GM-CSF) did not support proliferation of TF-1 cells

With the GM-CSF, TF-1 cells proliferated

Biological activity of hGM-CSF

Results of sugarcane and rice Sugarcane Out of 34 tested

plants, 22 showed unique hybridization patterns

Average tsp ~0.1%

Rice Out of 6 tested plants,

2 showed unique hybridization

Average tsp ~1.3%

Transgene silencing (Post-transcriptional gene silencing, PTGS)

Rapid mRNA turnover due to specific mRNA-destabilizing elements

Factors limiting sugarcane

Overall, the rice plants were found to have 1.3% tsp

This is 4-fold higher than the reported expression level in the seed of tobacco!

Also, we can achieve even higher levels of protein by employing a larger version of the Gt1 promoter

Conclusions

Hopefully we can successfully amplify this protein in sugarcane or rice seeds

Oral ingestion of hGM-CSF is not expected to have an immune response since these plants are typically ingested

Hopefully we can find a cheaper cost of production of this protein for clinical use

Help with humanizing breast milk for third world countries

Future plans

Sardana, Ravinder. et. al. 2007. Biologically Active Human GM-CSF Produced in the Seeds of Transgenic Rice Plants. Transgenic Research. 16: 713-721.

Wang, Ming-Li. et. al. 2004. Production of Biologically Active GM-CSF in sugarcane: a secure biofactory. Transgenic Research. 14: 167-178.

Blais, David R. et. al. 2007.Humanizing infant milk formula to decrease postnatal HIV transmission. TRENDS in Biotechnology. 25(9): 376-384.

References