[Screening and application of prokaryotic enhancer-like sequence 3A]

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Gene expression regulation is crucial for cellular function and therapeutic protein production.
• Prokaryotic systems offer efficient platforms for recombinant protein expression.

Purpose of the Study:

• To identify and characterize novel enhancer-like sequences from the Escherichia coli C600 genome.
• To construct an expression vector containing a prokaryotic enhancer-like sequence.
• To evaluate the impact of the identified enhancer on interferon gene expression and activity.

Main Methods:

• Screening of enhancer-like sequences using the chloramphenicol acetyl-transferase (CAT) gene as a reporter.
• Construction of a recombinant expression vector harboring the identified prokaryotic enhancer-like sequence.
• Expression and functional assay of interferon alpha-2b.

Main Results:

• A novel enhancer-like sequence, designated 3A, was isolated from E. coli C600, exhibiting distance and orientation independence.
• Sequence 3A demonstrated significant enhancement of reporter gene (beta-galactosidase) activity (7.11-fold in direct, 2.93-fold in reverse orientation), acting at the transcriptional level.
• An expression vector containing a functional region of 3A (3P3) increased interferon alpha-2b antiviral activity by 3.7-fold compared to the original plasmid.

Conclusions:

• The 3A enhancer-like sequence, identified from E. coli C600, effectively enhances gene expression.
• The developed expression vector utilizing the 3A enhancer facilitates high-level expression of interferon genes.
• This finding presents a valuable tool for improving the production of therapeutic proteins like interferon.