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Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’

Paper ID Volume ID Publish Year Pages File Format Full-Text
22714 43373 2016 10 PDF Available
Title
Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’
Abstract

•Streptavidin (SAV) production by an E. coli ‘leaky mutant’ was established.•Product secretion was improved by cultivation at 30 °C and Triton® supplementation.•bglA leader, constitutive bla promoter and full-length SAV gene were beneficial.•A bioreactor fed-batch process led to 2.6 μM of tetrameric SAV in 40 h (65.2 nM/h).•Less than 10% of biotin-blocked binding sites indicate a highly bioactive product.

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic ‘leaky mutant’ E. coli JW1667-5 (Δlpp-752:kan) at 30 °C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton® X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790 ± 166 nM were reached in shake flasks at a productivity of 49.6 nM h−1. Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608 ± 169 nM and a productivity of 65.2 nM h−1 in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3 ± 4.3% indicated a highly bioactive product.

Keywords
SAV, streptavidin; vvm, volume per volume and minuteEscherichia coli; Periplasmic leaky mutant; Chain length of streptavidin; Temperature; Secretion; Fed-batch cultivation
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Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 221, 10 March 2016, Pages 91–100
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