Unlike what your highschool bio teacher says — Biology can still happen without the living organism part

A guide to producing proteins Cell-Free Protein production of Immunglobulin A for insertion into infant formula

I’ve been researching breastmilk for a few months now, for the purpose of figuring out a way we can produce this extremely complex fluid outside of the human body, or at least synthesize some of it’s components.

Breastmilk contains around 2,500 different components. This is mostly water (87%), carbs, primarily lactose (7%), fats (4%), and proteins (1%). There are many different types of proteins, but antibodies are arguable the most important for the immune system.

Secretory Immunoglobulin A (sIgA) makes up about 90% of all antibodies in breastmilk. It acts as a barrier between the stomach lining. and bacteria that enters the body.

I started out by proposing the synthesis (production) of sIgA by using agrobacterium-mediated gene transfer to grow them in transgenic tobacco plants. That’s big words for giving tobacco plants instructions to produce the protein I want them to produce (in this case it was an antibody).

Although I proposed to use plants in this case because it was cheaper + more scalable, the production cost is still extremely high. It would be nearly impossible to lower the cost by 10x using that same process. As Astro Teller says, you can’t significantly decrease the process of something by 10% decrements.

So, I did a root cause analysis as to why this process was so expensive. About A major part of the process cost was protein purification (how the proteins are removed from the plant once they have been synthesized).

The cells need to kept alive and here is an image of the machinery that goes into that:

Although I worked around parts of this by using plants, growing plants is still quite expensive. The plants need to be kept at a constant temperature, given nutrients, maintained etc.

The cells also produce more than just the protein that I need, so they end up waisting a lot of the resources just for regular processes.

So then, I thought about how there’s only 4 primary components of the cell that are actually required in protein production. Why not isolate them, and synthesize proteins like that?

Turns out, there have been others who have thought of doing the same. This thought has been around for about 50 years, however, we only now are beginning to leverage it for protein synthesis. It’s not very widespread, but is very successful when used. By using this process, I can produce proteins at 10x less the cost and at about 28x the speed.

Now, just in case you’re curious, the rest if this article is the science behind how this can be done.

The central dogma of biology — Protein Synthesis

Here’s a quick simple bio lesson:

Transcription (in the nucleus of the cell)

1. RNA polymerase unzips the DNA that contains the gene for the protein to me made
2. Complimentary RNA nucleotides attach to one strand of the DNA. (Adenine bonds with thymine, and cytosine bonds with guanine)
3. The strand that is left is called mRNA

Translation (outside the nucleus of the cell)

1. The mRNA strand travels outside the nucleus into the cytoplasm
2. The tRNA molecules carrying specific amino acids attach to the mRNA molecules in a specific order.
3. The output are amino acids, which are ordered in a specific way and can now fold into a protein.

The old way of in vitro protein production

(this isn’t the new technology yet, just some context, so skip this too if you are just interested in the new way

Proteins are made of amino acids that fold in a certain way, and the instructions for the folding is encoded within DNA.

So, in order to make a cell produce a certain protein, we have to insert the DNA into that cell. There are a couple different ways that this is done, but the primary method is by using a plasmid, which are circular pieces of DNA carriers in bacteria. Bacteria is really really good at transferring DNA to other organisms, and plasmids are what is responsible for that.

Researchers came up with a way to leverage plasmids for DNA transfer. Genes of choice can be inserted into a plasmid, and that new structure (called a vector, since its now synthetic) is used to transfer DNA to a new organism

The vectors are then introduced to the cell, perform the gene transfer, and the cell begins to produce the protein encoded by the new DNA. Once the proteins reach a certain level of growth, the cells are lysed (a science word for tearing apart using a cool blender) and then purified, since there are more than just proteins in this substance.

But, there are several disadvantages to this process, including slow protein growth, expensive purification, toxic proteins, poor yields, high monitoring etc.

Cel-Free Protein Synthesis (SFPS)

(This is what everything’s been leading up to)

Cell lysates provide the correct composition and proportion of enzymes and building blocks required for translation. (Usually, an energy source and amino acids must also be added to sustain synthesis.) Cell membranes are removed to leave only the cytosolic and organelle components of the cell (hence the term, “cell-free extracts”). The first types of lysates developed for cell-free protein expression were derived from prokaryotic organisms. More recently, systems based on extracts from insect cells, mammalian cells and human cells have been developed and made commercially available.

Cell-free protein synthesis

Because there are really only a few components of the cell that operate to synthesize proteins, the entire cell isn’t required for this process.

There are two components required for in vitro protein expression:

1. The genetic material (mRNA or DNA) encoding the target protein (in this case its an antibody)
2. A solution that contains the molecules that will do the transcription and translation (from genetic material to protein). These include:

• RNA polymerases for mRNA transcription (the very first step)
• ribosomes for polypeptide translation
• tRNA and amino acids
• enzymatic cofactors and an energy source for the process
• in some cases other cellular components essential for proper protein folding

How these components are gathered:

1. Live cells are grown in liquid culture (just normal cell growth)
2. The cells are harvested out of the culture
3. The cells are busted open— usually done by vibrating the cells until they explode. Then gather the cellular components that are required for protein synthesis (as explained above).

Once the DNA is introduced to into the lysed cell, the process begins. About 2 hours later, the proteins can be extracted and purified.

There are several different types of cells that can be used for cell-free protein synthesis, each have advantages + disadvantages.

Secretory Immunoglobulin A is a higher order protein complex that is formed by several different parts that come together. One of the processes involved in this is called glycosylation, a process by which a sugar is attached to a part of the protein to help it properly fold.

There are only certain cells that are able to perform glycosylation — most of which are Eukorotic cells.

There is also the endoplasmic reticulum (ER), which is only present in Eukaryotic (animal) cells. The ER is responsible for part of the protein folding process — it creates an oxidative (lacking oxygen) environment, which helps form + stabilize disulphide bonds in the antibody.

The red lines show disulfide bonds.

Another protein, called BiP helps make sure that the heavy chain (at the bottom of the antibody) doesn’t unfold. Prolyl isomerases have also been proven to be involved in catalyzing the protein folding step.

Long story short, there are certain cellular components in mammalian cells to aid in the complex process of protein folding, so that it is done correctly with minimal errors. However, bacteria are extremely easy to use, fast to grow, need low maintenance and are easily manipulated.

That means that the next problem to be solved, is figuring out a way to generate a bacterial system that is optimized for the folding of eukaryotic proteins.

Open-cell Free Synthesis

So, smart people found a way to work around this problem as well, creating an open-cell free synthesis system, which allows us to add certain components to E. coli strains for improved protein folding.

This process has been done for Immunoglobulin G, where each of the following chaperones (components that can aid in protein folding) were tested to determine which improves expression the best.

Before expression IgA in a bacterial cell, a similar approach will take place to determine which chaperones should be over-expressed. Since high concentrations of purified chaperone proteins is extremely expensive and labor-intensive, we can instead can create bacterial strains that over-express the specific protein.

As I mentioned previously, bacteria contain plasmids, which are circular pieces of DNA. One the chaperones are determined, we can insert the over-expression gene into the plasmid of a bacteria, which will ensure that there are greater quantities of the specified proteins in the growing cell.

Even though the mammalian Endoplasmic Reticulum is a powerful system that can ensure proper protein folding, to increase efficiency, we can add substitutes to the cells. According to the research done on Immunoglobulin G, adding 3 of the top chaperones can improve protein folding by 40%.

A diagram of a comparison between regular protein synthesis, vs cell-free protein synthesis

The proteins can then be purified from the rest of the components. However, the there are only 5–7 cellular components that are filtered out, which decreases the number of times and ways the protein is to be purified.

And on that note, that’s the end of my thought process around cell-free protein synthesis. If you have any questions or thoughts, feel free to reach out, I’m more than happy to have a conversation!

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