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Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
24856 43541 2008 10 PDF Available
Title
Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli
Abstract

An alternative approach to the use of antibiotic selection markers for maintenance of recombinant plasmid vectors in Escherichia coli based on an aminoacid auxotrophy complementation has been developed. An E. coli M15-derivated glycine-auxotrophic strain of has been constructed by means of a PCR-based approach. This mutant strain contains a deletion in the glyA gene, which encodes for serine hydroxymethyl transferase, an enzyme involved in the main glycine biosynthesis pathway in E. coli. Also, we have constructed the complementation plasmid pQEαβrham derived from the commercially available expression vector pQE40 (QIAGEN) containing the glyA homologous gene under the control of the constitutive weak promoter P3. By using the E. coli M15ΔglyA strain combined with the pQEαβrham plasmid, a successful complementation system was achieved, allowing transformants to grow on minimal media without glycine supplementation. The capability of the new system E. coli M15ΔglyA/pQEαβrham for recombinant overproduction of rhamnulose 1-phosphate aldolase was evaluated in antibiotic free fed-batch cultures at controlled specific growth rate, obtaining high cell density cultures and high RhuA production and productivity levels comparable to those obtained with the conventional system. The new selection marker based on glycine-auxotrophy is a promising genetic tool, not only for recombinant protein production, but also for plasmid DNA production processes, where antibiotics can not be present in the medium formulation.

Keywords
Escherichia coli; Antibiotic-free plasmid selection system; Glycine auxotrophy complementation; Recombinant protein production; Rhamnulose 1-phosphate aldolase
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Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 134, Issues 1–2, 20 March 2008, Pages 127–136
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us