A small, high-copy-number vector suitable for both in vitro and in vivo gene expression

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Expression

Background:

• Expression vectors are crucial tools for recombinant protein production.
• Existing vectors may have limitations in expression efficiency or cloning flexibility.
• The development of novel vectors is essential for advancing biotechnological applications.

Purpose of the Study:

• To construct a novel high-copy-number expression vector, pUK.
• To evaluate the expression capabilities of the pUK vector for cloned genes.
• To enhance the cloning convenience of the vector's multiple cloning site.

Main Methods:

• Construction of the pUK vector by combining elements from pUC19 and pKK223-3.
• Cloning of chicken adenylate kinase (AK) and phospholipase A2 genes into the pUK vector.
• Assessment of in vitro gene expression using the Zubay coupled transcription/translation system.
• Evaluation of in vivo gene expression in Escherichia coli.

Main Results:

• The pUK vector was successfully constructed as a high-copy-number plasmid.
• High-level in vitro expression of AK and phospholipase A2 genes was achieved.
• High-level in vivo expression of the AK gene in E. coli was demonstrated.
• The multiple cloning site (MCS) of pKK223-3 was maintained, with AccI, BamHI, and SalI sites made unique for improved cloning.

Conclusions:

• The pUK vector is a versatile and efficient tool for high-level gene expression.
• pUK facilitates both in vitro and in vivo expression studies.
• The enhanced MCS of pUK offers greater cloning flexibility for researchers.