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Related Experiment Videos

High-level expression of human thymidylate synthase

J Pedersen-Lane1, G F Maley, E Chu

  • 1New York State Department of Health, Wadsworth Center, Albany 12201, USA.

Protein Expression and Purification
|July 1, 1997
PubMed
Summary

This study presents a novel method for high-level expression of human thymidylate synthase (TS) in E. coli by modifying its cDNA sequence. This approach significantly enhances protein production for research applications.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Protein Expression

Background:

  • Human thymidylate synthase (TS) is a crucial enzyme in DNA synthesis.
  • Previous methods achieved low expression levels of recombinant TS.
  • Understanding factors influencing TS gene expression is vital for biotechnological applications.

Purpose of the Study:

  • To develop a method for significantly increasing the expression of human thymidylate synthase (TS) in Escherichia coli.
  • To identify the genetic modifications responsible for enhanced TS protein production.
  • To compare the expression and activity of modified TS with native TS.

Main Methods:

  • Modification of thymidylate synthase (TS) cDNA sequence, specifically altering purine bases in early codons to thymine.

Related Experiment Videos

  • Expression of modified and unmodified TS cDNA in Escherichia coli.
  • Purification of recombinant TS enzyme.
  • In vitro transcription and translation assays using a rabbit reticulocyte system.

    • Main Results:

    • Achieved 25-30% protein expression of human TS in E. coli, yielding 200-400 mg per 2-liter culture.
    • Unmodified TS cDNA expression was only 0.1-0.2% of total cellular protein.
    • In vitro translation of native TS mRNA was reduced 20-fold compared to modified TS mRNA, indicating a translational block.
    • Modified TS showed comparable activity to native TS, despite N-terminal differences.

    Conclusions:

    • The GC content and sequence of the 5' coding region significantly impact TS gene expression in both prokaryotic and eukaryotic systems.
    • Codon optimization of the TS gene is a key strategy for achieving high-level protein expression.
    • This method provides a robust system for producing large quantities of active human TS for further study.