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Updated: Mar 14, 2026

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Published on: November 1, 2019

A Positive Selection for Nucleoside Kinases in E. coli.

Nirav Y Shelat¹, Sidhartha Parhi², Marc Ostermeier²

¹Chemical Biology Interface Graduate Program, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, United States of America.

|September 28, 2016

Summary

We engineered a method to select for nucleoside kinase activity in E. coli by moving the kinase to the periplasmic space. This approach overcomes toxicity and localization issues, enabling better screening of engineered kinases and prodrugs.

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Published on: March 16, 2011

Area of Science:

Biotechnology
Molecular Biology
Enzyme Engineering

Background:

Engineering heterologous nucleoside kinases in E. coli is challenging due to nucleoside roles in cell processes.
Metabolic activation of nucleoside analogs can be toxic, disrupting DNA replication and transcription.
Endogenous kinases can mask engineered kinase activity, complicating assessment.

Purpose of the Study:

To develop a positive selection method for discerning heterologous nucleoside kinase activity.
To overcome toxicity and localization issues associated with previous selection schemes.
To create a general strategy for selecting or evaluating nucleoside kinases.

Main Methods:

Engineered E. coli with a knockout of PhoA alkaline phosphatase.
Translocated herpes simplex virus thymidine kinase (HSV-TK) to the periplasmic space using a tat-signal sequence.
Utilized the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) by HSV-TK for selection.

Main Results:

Functional HSV-TK was successfully translocated to the periplasmic space.
Periplasmic HSV-TK conferred significant resistance to the toxic nucleoside analog AZT.
Resistance decreased dramatically above 40 μg/ml AZT, indicating a selection threshold.

Conclusions:

The periplasmic translocation strategy enables selection for functional nucleoside kinases.
This method overcomes toxicity and localization challenges in E. coli.
The strategy is applicable for selecting higher activity kinase variants and evaluating prodrugs.