Challenges in Producing Antibodies with Plants

Scientists are looking for ways to produce proteins, like antibodies, using plants. One popular plant for this is Nicotiana benthamiana, which helps make proteins in a process called agroinfiltration. However, sometimes the plants can break down these proteins, making it hard to get enough of them. This article discusses the challenges faced when using N. benthamiana to produce a specific HIV-neutralizing antibody called 2F5.

#The Problem with Protein Breakdown

When proteins are made in N. benthamiana, natural plant enzymes known as Proteases can cause problems. These enzymes can cut the proteins into smaller pieces or even ruin them. For example, the HIV antibody 2F5 is often damaged by these enzymes when it is produced in the plant. In earlier research, it was found that the enzyme SBT5.2a is one of the main culprits responsible for this damage. When a protease inhibitor called PMSF was added, it stopped the breakdown of the antibody.

#The Role of SBT5.2a

SBT5.2a is a specific type of protease found in the fluids of N. benthamiana. After further studies, it was shown that this enzyme can cut the antibody 2F5 in a specific part of its structure. To see if SBT5.2a is necessary for cutting 2F5 in the plant fluids, researchers conducted tests. They used a special tagging method to visualize the antibody and confirmed that the tagged version broke down just like the untagged one. When using PMSF or another protease inhibitor, the breakdown of the antibody stopped, confirming that SBT5.2a is important in this process.

#Investigating the Action of SBT5.2a

To learn more, researchers used a technique called Virus-induced Gene Silencing (VIGS) to block the SBT5.2a gene in N. benthamiana. When they did this, they found that plants lacking this gene could not break down the 2F5 antibody. This was a clear indication that SBT5.2a is needed for the antibody's breakdown.

#Genome Editing to Confirm Findings

In another approach, researchers used CRISPR technology to make changes to the plant DNA, effectively knocking out all three genes that produce SBT5.2a. The resulting plants behaved similarly to normal plants but lacked the active proteases that could break down the antibodies. Tests showed that these modified plants could keep 2F5 intact when compared to normal plants, suggesting that removing SBT5.2a could help increase the amounts of 2F5 produced.

#Accumulation of Antibodies

Research showed that in plants with the SBT5.2a enzymes removed, the total amount of 2F5 was three times greater than in normal plants. This finding was exciting, as it suggested that knocking out SBT5.2a might boost the production of antibodies in plant systems, which is crucial for drug development.

#Testing for Secretion

To find out if the 2F5 antibody was being secreted into the plant fluids, researchers did further tests. They co-expressed the 2F5 antibody alongside a fluorescent protein to track where it went. The results showed that only a small percentage of the antibody was detected in the fluids, while a larger amount of another protein was found. This raised questions about whether antibodies are actually secreted from these plants.

#Comparing Antibody Distribution

The majority of the antibody 2F5 remained in the plant’s cells rather than being released into the fluids, which is different from what many expected. Typical belief was that antibodies should be secreted into fluids, but the findings suggested that 2F5 may not be. Earlier studies had also noted low amounts of antibodies found in plant fluids, indicating this is not an isolated issue.

#Possible Reasons for Retention

Researchers speculated about why the 2F5 antibody stayed inside the plant cells instead of being secreted. It has been suggested that there might be signals in the antibody’s structure that keep it from leaving the cells. Additionally, chaperone proteins may bind to the antibodies and prevent them from being secreted.

#Conclusion

This research highlights important challenges and possibilities in using N. benthamiana for producing therapeutic proteins, especially antibodies like 2F5. By removing certain proteases, it was possible to increase the amount of antibody produced, showing that plant systems can be optimized for better yield. At the same time, it called into question the common belief that antibodies are primarily secreted from plants.

Overall, this study opens new avenues for improving the production of proteins in plants and enhances our knowledge about how these proteins behave within plant systems. It reinforces the idea that understanding how to manage and manipulate plant enzymes is vital for successful protein production, especially in the context of developing therapies against diseases like HIV. As researchers continue to explore these methods, they hope to refine the process for better outcomes in protein expression using plants.