Periplasmic expression in and release of Fab fragments from <i>Escherichia coli</i> using stress minimization

Hsu, Chia-Chang; Thomas, Owen R.T.; Overton, Tim W.

doi:10.1002/jctb.4672

Cited by 25 publications

(23 citation statements)

References 25 publications

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“…Data obtained from the scFv and DNA concentrations in the culture media ( Fig. 3 ) show that both have relatively the same behavior so wherever scFv concentration rises subsequently DNA concentration is increased which demonstrates that the produced soluble scFv in the media is resulted by the cell lysis 13 hence that is not a favorable product because of cytoplasmic and no oxidized scFv protein contaminations. Therefore, the responses related to the concentration of scFv in the culture media were neglected.…”

Section: Resultsmentioning

confidence: 91%

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Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Mesgari-Shadi¹,

Sarrafzadeh²

2017

Bioimpacts

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Introduction: Single chain variable fragment (scFv) antibodies are reduced forms of the whole antibodies that could be regarded as an alternative tool for diagnostic and therapeutic purposes. The optimization of processes and environmental conditions is necessary to increase the production yields and enhance the productivity. This can result in a cost-effective process and respond to the high demand for these antibodies. Methods: In this research, physical and chemical factors influencing the batch fermentation was investigated in 50 mL batch tubes using minimum media to find the optimum conditions for production of a single chain variable fragment antibody in the Escherichia coli HB2151. Experimental designs were used to screen the effective parameters and to optimize the main factors. Results: Arabinose was used instead of IPTG as a cheaper and nontoxic inducer and its optimum concentration was determined 0.1% (w/w). Induction duration time and filling volume fraction were set on the relatively better states 24 hours and 1/10 respectively. Regarding our previous study, stationary phase of the cell growth was selected as induction start time that showed higher specific scFv production yields (YP/X) in the minimum media. Finally, a statistical experimental design was extended to a central composite design (CCD) and analysis was performed based on sucrose and sorbitol concentrations producing osmotic condition for induction. The optimum region in the contour plot for the periplasmic scFv production was an osmotic circle area with total sugar molarity 0.8 to 0.9. Conclusion: Sugars such as sucrose and sorbitol producing osmotic conditions could lead to periplasmic scFv concentrations up to 2.85 mg/L of culture media improving scFv concentration near to five times of the average of the screening step (0.59 mg/L).

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Section: Resultsmentioning

confidence: 91%

“… 9 - 12 Metabolic considerations could be an effective approach to influence the induction process toward higher recombinant protein production. 13 …”

Section: Introductionmentioning

confidence: 99%

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Mesgari-Shadi¹,

Sarrafzadeh²

2017

Bioimpacts

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“…Despite the drop in scFv accumulation when compared to scFv::Bla, the screening system nonetheless generated signal peptides capable of better production of scFv 13R4 than PelB sp . Future work should focus on further development of the system, including fermentation development to optimise process conditions and minimise the quantity of scFv in the culture medium for selected signal peptides 17 .…”

Section: Resultsmentioning

confidence: 99%

“…These requirements for the design of periplasmic RPP are additional to the requirements for any RPP process, such as balancing the metabolic requirements of RPP and growth to prevent metabolic burden or the stringent response, and prevention of excessive protein misfolding that would trigger the heat shock response (reviewed by 2 ). Therefore signal peptide choice is one element of process design, along with optimisation of fermentation conditions 17 .…”

Section: Introductionmentioning

confidence: 99%

Development of a generic β-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli

Castiñeiras

Williams²,

Hitchcock³

et al. 2018

Sci Rep

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Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 β-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. β-lactamase was fused to the C-terminal of scFv and β-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for β-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.

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“…It is noticed that there are many bands in the excreted fraction in Fig. 5 a, this might because the metabolic stress in bacterial generating recombinant proteins such as secreted proteins can cause cell lysis during the latter fermentation [ 38 ]. The anti-thrombin activity of HV3 was used to illustrate the difference of target proteins before and after the removal of the Ffu fusion system.…”

Section: Discussionmentioning

confidence: 99%

A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli

Cheng

Cui

et al. 2017

Microb Cell Fact

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BackgroundThe high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner.MethodsBased on the modified Wilkinson and Harrison algorithm and the preliminary verification of the solubility-enhancing ability of β-FFase truncations, three β-FFase truncations (i.e., Ffu209, Ffu217, and Ffu312) with a native signal peptide were selected as novel Ffu fusion tags. Four difficult-to-express protein models; i.e., CARDS TX, VEGFR-2, RVs and Omp85 were used in the assessment of Ffu fusion tags.ResultsThe expression levels and solubility of each protein were markedly enhanced by the Ffu fusion system. Each protein had a favorable Ffu tag. The Ffu fusion tags performed preferably when compared with the well-known fusion tags MBP and NusA. Strikingly, it was confirmed that Ffu fusion proteins were secreted into the periplasm by the periplasmic analysis and N-amino acid sequence analysis. Further, efficient excretion of HV3 with defined anti-thrombin activity was obtained when it was fused with the Ffu312 tag. Moreover, HV3 remained soluble and demonstrated notable anti-thrombin activity after the removal of the Ffu312 tag by enterokinase.ConclusionsObservations from this work not only complements fusion technologies, but also develops a novel and effective secretory system to solve key issues that include inclusion bodies and degradation when expressing heterologous proteins in E. coli, especially for proteins that require disulfide bond formation, eukaryotic-secreted proteins, and membrane-associated proteins.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0845-z) contains supplementary material, which is available to authorized users.

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Periplasmic expression in and release of Fab fragments from Escherichia coli using stress minimization

Cited by 25 publications

References 25 publications

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Osmotic conditions could promote scFv antibody production in the Escherichia coli HB2151

Development of a generic β-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli

A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli

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