Enabling microbial production of a human enzyme

gilishklar
Oct 25
2 min read

Scala’s underlying computational design enables bacterial production of a complex human enzyme

Why it matters

Producing complex human enzymes in bacteria is a major hurdle for biotechnology, since many proteins are too unstable to fold and function outside human cells. Human acetylcholinesterase (AChE) is one such enzyme and resisted all attempts at bacterial production for more than 20 years. Scala’s stability design technology overcame this barrier, enabling high-yield production of active AChE in bacteria. This breakthrough transformed a protein long considered intractable into a practical platform for structural studies, enzyme engineering, and therapeutic development.

Problem

AChE is a large human enzyme of more than 540 amino acids, with its catalytic site buried deep inside a long narrow channel. It is heavily glycosylated and stabilized by multiple disulfide bonds, making it impossible to produce in bacteria using conventional methods. This prevented its development for potential therapeutic and biodefense applications, such as detoxifying organophosphate compounds.

What was done

In a single design round, Scala’s underlying stability design technology was applied to AChE to generate five variants, each carrying 17–67 mutations. The designs were selected to improve folding and stability while preserving the enzyme’s catalytic site and overall activity. The variants were then produced and tested in bacteria.

Results

Design scope: Five AChE variants were generated in a single design round, each carrying 17–67 mutations.
Expression and activity: All variants expressed as soluble, active enzymes in E. coli; wild type showed no functional expression.
Thermal stability: The best variant displayed a +20 °C increase in melting temperature relative to wild type.
Catalytic performance and structure: Catalytic efficiency and active-site geometry were identical to wild type, and the stabilized enzyme crystallized readily.

Impact

This project demonstrated that even the most complex human enzymes can be redesigned for efficient bacterial production without loss of function. By converting an intractable protein into a workable system, Scala enabled structural biology, large-scale studies, and further engineering campaigns. The success with AChE illustrates how stability design can unlock enzymes of medical and therapeutic importance that were previously out of reach.

Data highlights

Expression/activity panel: Wild-type AChE showed no functional expression in E. coli, while all five designed variants were soluble and enzymatically active.

Thermostability panel: Differential scanning fluorimetry revealed up to a 20 °C increase in melting temperature in the best variant.

(Crystal structure panel) X-ray crystallography confirmed the stabilized AChE retained native active-site architecture.

References

Goldenzweig A, Goldsmith M, Hill SE, et al. Automated structure- and sequence-based design of proteins for high bacterial expression and stability. Mol Cell. 2016;63(2):337–346. doi:10.1016/j.molcel.2016.06.012