Indrduction

Good afternoon everyone! I hope you’ve been having a great day so far!

The subject of today’s presentation is ‘Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice’.

The main points I will be talking about today are firstly HSA and its market demand, secondly rice as an attractive bioreactor for mass production of rHSA and the controversy that this fact brings, next I’ll concentrate on the methods of obtaining selectively terminable transgenic rice line expressing HSA in rice seeds and a process for recovery and purification. Finally, we’re going to look at the results and I believe we’ll have a decent discussion.

HSA

Let’s begin with a short description of Human Serum Albumin.

Human serum albumin is the most abundant protein in human blood plasma and is unique from other blood proteins as it’s not glycosylated. It is produced in the liver. Albumin constitutes about 50% of the blood serum protein. It is soluble, globular and monomeric. The reference range for albumin concentrations in serum is approximately 35 - 50 g/L.

It has a serum half-life of approximately 20 days. It has a molecular mass of 67 kDa.

I’m gonna move now to HAS’s functions in the body as well as its clinical use.

Albumin transports hormones, fatty acids, and other compounds, buffers pH, and maintains osmotic pressure.

HSA is widely used to treat severe hypoproteinemia and hyperbilirubinemia, post-surgery and post-traumatic shock, and hepatocirrhosis (hepatocirołsis)

HSA is also frequently used in biochemical applications, such as the formulation of protein therapeutics, cell culture media, drug delivery, cryopreservation, in vivo diagnostics, vaccine formulation and manufacturing, and infertility treatments. Many other novel uses of HAS in biological applications have been explored.

So as u can see there is a huge demand for HSA nowadays. The market demand for HSA is estimated at more than 500 tons per year worldwide. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors, like human immunodeficiency virus (HIV) and hepatitis.

In fact, illegal plasma collection has caused HIV to spread rapidly, creating what are known as AIDS villages in Henan Province in China .It was reported that the shortage of human plasma led to a rapid increase in price of HSA, which in turn resulted in a brief rise in the number of fraudulent albumin medicines on the market.

Just to give you an idea how much pHSA costs Ive googled an example. And as u can see the prices r extremely high!

Here is a question: How to meet current and future demand for HSA when we can’t rely on blood donations?

The development of a low cost method for the production of recombinant HSA (rHSA) is essential as a safer and potentially unlimited alternative to plasma HSA (pHSA). Scientist have been dealing with this fact for years.

Over the past decades, various expression systems have been used to produce rHSA, including Escherichia coli , Saccharomyces cerevisiae , Pichia pastoris, transgenic animals. Various genetically modified plants have been explored to express heterogeneous HSA, including potato, tobacco and rice.

Although rHSA has been successfully expressed in these systems, only rice has proven to be cost-effective at large scale. Why?

Rice grains are promising vehicles for producing recombinant proteins, because they can achieve high accumulation of recombinant protein, display high levels of protein stability, stored for long periods of time, and are well controlled on a production scale.

Expressing HSA in rice makes us define this plant as GMO what stands for Genetically Modified Organism. Here comes the controversy!!!

The major concern about using rice to produce rHSA is unintentional spreading of the transgene into the food chain. We have to mention here that rice is a major world food staple.

Although physical containment measures

(isolation zones, buffer zones, fencing, etc.) have been taken to contain transgenes, incidents of transgene escape have still occurred.

What if it is possible to grow transgenic rice in order to produce rHSA and prevent the transgens from spreading into regular rice?

No method has yet been reported that involves using chemical traits to prevent transgenes from spreading into regular rice, and which may be applicable to an rHSA bioreactor system. Therefore, a simple and reliable containment method is highly needed from a biosafety point of view.

Chinese scientists have developed a transgenic rice expression system for rHSA with a novel built-in transgene containment technology as well as the process of recovery and purification.

Materials & Methods

Now let me move on to what was what was done, what materials and methods were used in this study.

Scientist started with a vector construction.

The resulting binary vector for rice transformation consist of the HSA expression, glyphosate resistance, and RNAi cassettes, responsible for benzaton sensitivity.

Ben-ta-zon is a selective herbicide as it only damages plants unable to metabolize the chemical

Glyphosate  is a broad-spectrum systemic herbicide used to kill weeds, especially annual broadleaf weeds and grasses known to compete with commercial crops grown around the globe.

This T-DNA transformation construct was introduced into Agrobacterium tumefaciens using an electroporator and than a local rice was transformed. Glyphosate was used as the selection agent.

Plants of transgenic rice and untransformed rice were grown in a solution in the greenhouse. Plants were tested for the glyphosate tolerance and benzaton sensitivity.

Scientist analyzed protein by SDS-PAGE and the quantification of r HSA was determined using Elisa Human Albumin Quantification Kit.

All the purification steps were based on fast protein liquid chromatography experiments (FPLC).

Further studies determined molecular weight, N-terminal amino acid residue and protein conformation.

Molecular weight: MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight analysis)

N-terminal amino acid residue determination: Edman degradation/sequencer

Protein conformation: CD spectra for pHSA and rHSA

RESULTS

Now I’d like to take a look at the results.

209 independent transformed events were generated and grown in the field to harvest seeds.

In the first generation line named HSA – 84 was found as the one of highest expression lines. That was examinated using SDS – PAGE. (figure) .

rHSA was estimated to account for 0.72% of the total dry weight of the dehusked rice seeds.

What is more the transgenic rice seeds have a different phenotype in comparison with no transgenic control seeds. Due to the visual difference it was easy to determine the transgenic seeds and non transgenic ones. The terminable transgenic rice shown no visible difference in growth and development compared to non – transgenic.

It was proved as well that the protein was stably expressed in different generations.

As I mentioned during the vector construction description the genetic cassettes for HSA expression, RNAi of the bentazon detoxifying enzyme, and glyphosate tolerance were all in a single T-DNA fragment so the chance of separation among these three cassettes was extremely low and this fact can explain the stable expression.

The sensitivity to benzaton and glyphosate was tested on the plants of 3^rd generation HAS-84 what is picturised here.(figure). One spray of benzaton killed 100% of transgenic plants, but non of the non – transgenic rice within 10 days. In contrast 1 spray of glyphosate killed all of the conventional rice plants in 10 days, but it didn’t affect any transgenic plants as it was expected. Moreover the similar results were observed after tests on the plants from 4^th and 5^th generation what indicates that the sensitivity to those two herbicides was stably inherited in HAS – 84 line

Purification scheme

In this study scientist developed a purification scheme which includes crude extract pretreatment and two chromatography steps.

As the initial step Thermal treatment was used to inactivate proteases and denaturate some rice seed storage proteins. The other unwanted proteins were removed by precipitation at pH =4.5. the recovery rate after this step was 90.5%.

Low molecular weight proteins were removed from the supernatant

By using the 1^st chromatography with ANX Sepharose Fast Flow column. And if we look at the table nw we can notice a huge difference in a purity (nearly 30%)and recovery of 85.8%.

After the Buthyl Highh Performance column was used to remove the rest of non – HSA proteins.

The rHSA eluted from Buthyl HP column appeared as a single band of pure protein in SDS-PGE.

The total recovery after the protein purification steps was 62.4%

What is more the final purity of rHSA was comparable to that of pHSA control with the purity of 99%.

Futher studies were performed to check the similarity of rHSA to the natural protein.

The mass/charge value showed that rHSA has equivalent molecular weight of 66441Da which is very close to the 66531 Da that natural protein has.

The N- Terminal amino acid residues were determined as DAHKSEVAHR.

The result shown that the rHSA was identical to the pHSA in the primary structure, but the primary structure does not inform us that expressed protein is functional and active.

In order to confirm that the recombinant protein had the same conformation as the natural one the CD spectrum analysis was done. As shown at the figure the spectrum of rHSA is nearly identical to the pHSA.(fig 7)

Disscusion

Well, that brings us to the end of the final section. Now, I’d like to briefly summarise.

What is extraordinary in presented paper is the fact that scientist managed to develop a transgenic line for high expression of rHSA with a built in spread control technology.

If those plants spread accidently into regular rice they could be killed by benzaton during regular weed control, so we can easily say that described method is inexpensive and convenient. Sophisticated equipment is not required and obtaining rHSA could be easily scaled up planting larger area of transgenic rice. Another objective of this stude was to develop a a good practice scheme for isolating rHSA of high purity. I think I have said a lot about it few minutes before so that I’ll just remind you that the final product reached 99% purity which is pure enough to be used as a cell culture component. It was proved as well that the rHSA produced by the transgenic lines is identical to native protein from blood, so that scientist expect that the rHSA protein that they obtained is biologically identical to its native protein.

However more generations of transgenic plants still need to be monitored as the long term heritability of the RNAi has not yet been well studied

Scientist believe that recombinant protein production can be increased to kilogram scale, but that needs further studies, as well.

• That brings the presentation to an end. I hope you didn’t get bored at all and you enjoyed the subject as much as I did.

• If anyone has any questions, please feel free to ask them and I’ll do my best to answer.