Production of Reverse Transcriptase and DNA Polymerase in Bacterial Expression Systems

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• DNA amplification and reverse transcription enzymes are critical for diagnostics and research.
• High-level production of these enzymes is essential for their widespread application.
• Escherichia coli is a common host, but challenges exist in recombinant protein production.

Purpose of the Study:

• To evaluate Vibrio natriegens as an expression host for mutant Taq DNA polymerase and mutant M-MLV reverse transcriptase.
• To compare enzyme production levels and solubility in V. natriegens versus Escherichia coli.
• To investigate extracellular enzyme production in V. natriegens.

Main Methods:

• Gene expression of mutant Taq DNA polymerase and M-MLV reverse transcriptase in V. natriegens and E. coli.
• Optimization of expression conditions.
• Analysis of intracellular and extracellular protein production using percentage of total cell proteins (TCPs).
• Assessment of protein solubility.

Main Results:

• Intracellular M-MLV production: 11% TCPs in V. natriegens vs. 16% TCPs in E. coli; soluble protein: 11% in V. natriegens vs. 22% in E. coli.
• Intracellular Taq pol production: 30% TCPs in V. natriegens vs. 26% TCPs in E. coli; soluble protein: 23% in V. natriegens vs. almost non-soluble in E. coli.
• Substantial extracellular Taq pol production was detected in V. natriegens.

Conclusions:

• Vibrio natriegens is a viable alternative host for recombinant enzyme production.
• V. natriegens demonstrates potential for high-level soluble production of Taq DNA polymerase and M-MLV reverse transcriptase.
• Extracellular production of Taq pol in V. natriegens offers additional production possibilities.