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Related Concept Videos

Gene Therapy00:59

Gene Therapy

Gene therapy is a technique where a gene is inserted into a person’s cells to prevent or treat a serious disease. The added gene may be a healthy version of the gene that is mutated in the patient, or it could be a different gene that inactivates or compensates for the patient’s disease-causing gene. For example, in patients with severe combined immunodeficiency (SCID) due to a mutation in the gene for the enzyme adenosine deaminase, a functioning version of the gene can be inserted. The...
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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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The Central Dogma01:20

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Updated: Jun 14, 2026

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells
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In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Published on: November 13, 2014

A new method for gene synthesis and its high-level expression.

Shi-shu Cao1, Zhi-qiu Hu

  • 1Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA. caoshishu@yahoo.com

Journal of Microbiological Methods
|September 8, 2009
PubMed
Summary
This summary is machine-generated.

Chemically synthesized Citrobacter braakii phytase (appA-c) was heterologously expressed in Pichia pastoris, achieving the highest reported phytase activity. This optimized gene expression method offers potential for broader applications in enzyme research.

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Molecular Biology

Background:

  • Phytases are crucial enzymes for phosphorus release from phytate.
  • Efficient heterologous expression of phytase genes is essential for industrial applications.
  • Citrobacter braakii phytase (appA) offers potential for improved enzyme properties.

Purpose of the Study:

  • To chemically synthesize and optimize the Citrobacter braakii phytase gene (appA-c).
  • To achieve high-level heterologous expression of appA-c in Pichia pastoris.
  • To characterize the biochemical properties of the recombinant phytase.

Main Methods:

  • Chemical synthesis of appA-c gene using oligonucleotides and overlap PCR.
  • Cloning into Pichia pastoris expression vector pPIC9 under AOX1 promoter control.
  • High-throughput screening of transformants for phytase activity.
  • Fermentation in a 5 L bioreactor and enzyme characterization.

Main Results:

  • Successful synthesis and cloning of appA-c.
  • Identification of transformants with high phytase activity.
  • Secretion of 12,116 U/ml phytase activity in Pichia pastoris culture supernatant.
  • Optimal activity at pH 4.0 and 50°C, with specific activity of 3147 U/mg.
  • Determined kinetic parameters (Km = 0.5 mM, Vmax = 3085 U/mg) for sodium phytate.

Conclusions:

  • The study reports the first successful heterologous expression of a chemically synthesized Citrobacter braakii appA gene.
  • Achieved unprecedentedly high phytase expression levels and activity in Pichia pastoris.
  • The novel gene optimization and synthesis strategy is applicable to other enzyme research.
  • This work provides a robust platform for producing highly active phytase for various applications.