# Bacterial Production of CDKL5 Catalytic Domain: Insights in Aggregation, Internal Translation and Phosphorylation Patterns

## TL;DR

This study explores how to produce a key part of the CDKL5 protein in bacteria, revealing insights into its structure and function.

## Contribution

The study introduces a novel method to produce a soluble and active CDKL5 catalytic domain in bacteria, enabling comparative studies with eukaryotic-expressed proteins.

## Key findings

- The CDKL5 catalytic domain predominantly aggregates in E. coli but can be made soluble with solubility tags and optimized conditions.
- The bacterial-produced CDKL5 is hypophosphorylated compared to its eukaryotic counterpart.
- The findings provide a platform for biophysical studies of CDKL5 and its role in neurodevelopmental disorders.

## Abstract

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K+ and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction.