Extracellular Secretion System Could Solve Industry Endonuclease Production Challenges

Endonuclease enzymes play a vital role in modern biopharmaceutical production, yet manufacturing them in bulk using traditional E. coli systems is challenging. New research suggests secretory expression could be a more effective alternative.

In biopharmaceutical manufacturing, endonucleases are used to digest any unwanted DNA and RNA that is released when the host cells of the expression system are lysed during the protein harvesting process.

The problem for manufacturers is that endonucleases have the same effect in the cells in which they are produced, says lead study author Ramakrishna Vadde, PhD, a professor from Yogi Vemana University in Andhra Pradesh, India.

“It is very difficult to express recombinant endonucleases using the bacteria Escherichia coli as the expression system,” he tells GEN. “This problem is mainly attributed to the fact that both DNA and RNA, the natural substrate molecules of these enzymes, play an important role in the growth and survival of the host bacteria themselves.”

A further complication is that endonuclease production puts a heavy burden on host cell metabolism. Vadde says, “Overexpression of these proteins may impair cellular processes such as proper protein folding, leading to protein aggregation, inclusion body formation, and enhanced protein degradation by host cell proteases.

“These combined challenges make recombinant endonuclease production in E. coli technically demanding and require carefully optimized expression and purification strategies.”

Extracellular secretion

A potential solution—presented in the new study—is to use an expression system that ensures the endonucleases are transported outside the cell, where they cannot degrade any host genetic materials.

Vadde says, “Our solution is based on the use of a proprietary secretion-based expression platform, BacSec, which directs the recombinant nuclease out of the cytoplasm and into the extracellular medium during production.

“By physically separating the enzyme from the host cell’s genomic DNA, plasmid DNA, and RNA, the risk of intracellular nucleic acid degradation is significantly minimized. This allows the production host to maintain normal growth, replication, and protein synthesis while expressing the endonuclease at high levels.

In addition to reducing toxicity, extracellular secretion offers several manufacturing advantages, according to Vadde, who cites reduced contamination risk as an example.

“The enzyme is produced directly in the culture supernatant, eliminating the need for cell disruption and reducing contamination from host-cell proteins and intracellular components. This simplifies downstream purification, improves product recovery, and lowers production costs. Furthermore, secretion often promotes proper protein folding and activity, avoiding the challenges associated with inclusion body formation and complex refolding procedures commonly encountered with intracellular expression of toxic proteins,” he says.

The approach can also reduce the cost of goods sold (COGs), according to Vadde, who adds, “Our platform enables the cost-effective production of active recombinant endonucleases such as Serratia marcescens endonuclease and bovine DNase I while overcoming the host-cell toxicity limitations that have traditionally hindered their manufacture.

“It represents a significant commercial opportunity, as these enzymes are essential consumable reagents used routinely by biologics manufacturers, vaccine developers, gene therapy companies, cell therapy producers, and contract development and manufacturing organizations (CDMOs) worldwide,” he says.

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