Characterization of bacteriophage λ Q− mutant for stable and efficient production of recombinant protein in Escherichia coli system

✍ Scribed by Chan-Shing Lin; Bor-Yann Chen; Tai Hyun Park; Henry C. Lim

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 135 KB
• Volume: 57
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19980305)57:5<529::aid-bit4>3.0.co;2-i

✦ Synopsis

We previously demonstrated that the system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S -mutant was only twice as high as that of the single copy. To increase the expression in the system, we constructed a Q -mutant vector that can be used in long-term operations such as a two-stage continuous operation. The Q -mutant phage is deficient in the synthesis of proteins involved in cell lysis and DNA packaging, while the S - mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q -DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The ␤galactosidase expression per unit cell by the Q -mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S -mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q - mutant from the lysogenic state to the lytic state for the maximum production of ␤-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation.