Cytosolic expression of functional Fab fragments in <i>Escherichia coli</i>
 using a novel combination of dual SUMO expression cassette and EnBase<sup>®</sup>
 cultivation mode

Rezaie, Faegheh; Davami, Fatemeh; Mansouri, Kamran; Amiri, Sara; Fazel, Ramin; Mahdian, Reza; Davoudi, Nooshin; Enayati, Somayeh; Azizi, Mohammad; Khalaj, Vahid

doi:10.1111/jam.13483

Cited by 15 publications

(8 citation statements)

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“…Furthermore, hydrophobic reactions that make inclusion bodies and aggregation of the recombinant proteins happen less (Krause et al, 2010) and for this reason a higher soluble form of α-luffin was achieved at 30 °C and 25 °C in comparison to 37 °C. On the other hand, the chaperone-like manner of SUMO tag and high activity of chaperones at 30 °C can improve the solubility rate of α-luffin (Rezaie et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

et al. 2018

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α-Luffin, found in Luffa cylindrica seeds, is a type I ribosome inactivating proteins. Cytotoxic effects make it an appropriate candidate for the construction of immunotoxins and conjugates. Because of limited natural resources, recombinant technology is the best approach to achieve large-scale production of plant-based proteins. In the present study, α-luffin protein was expressed in E. coli and the effects of different temperature conditions, SUMO fusion tag, and cultivation strategies on total expression and solubility were investigated. Protein expression was evaluated at different intervals (0, 4, 6, 8, 24 h) postinduction. Our results showed that EnBase had higher efficiency than LB, and maximum solubility and total protein expression were achieved 24 h after induction at 30 °C and 25 °C, respectively. It was shown that SUMO tag is an effective strategy to improve protein solubility.

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Section: Discussionmentioning

confidence: 99%

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

et al. 2018

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“…Considering our protein characteristics besides EnBase system special features, 30 and 25 °C temperatures were studied. The 30 °C was chosen based on recommended EnBase protocol due to higher oxygen solubility and reduced evaporation while studying usual nontoxic proteins expression ( 25 , 43 ). Here also, this temperature resulted in higher specific production of total protein in both batch and fed-batch method ( Figs.…”

Section: Discussionmentioning

confidence: 99%

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

et al. 2018

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BackgroundThe increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.ObjectivesHere, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.Materials and MethodsThe encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.ResultsIn fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.ConclusionsOur founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.

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“…In the Studier's autoinduction medium [84], the growth is supported at the beginning by glucose, and when glucose is exhausted protein expression is autoinduced by a diauxic shift to lactose utilization, while glycerol is also coutilized as a major carbon source during expression. The most recent EnBase ® medium (EnPresso, GmbH, Berlin, Germany), in which glucose, as a primary carbon source, is gradually provided from a soluble polysaccharide by biocatalytic degradation [85], has been successfully used for high-yield cytoplasmic and periplasmic expression of several proteins including antibody fragments [86][87][88][89]. Several studies have demonstrated that the yield of recombinant antibody fragments in E. coli significantly improves at growth temperature below 30 • C, likely due to the reduction of translation rate that favors proper Ig-like folding and reduces aggregation [90][91][92].…”

Section: Overcoming Drawbacks In Small-scale Productionsmentioning

confidence: 99%

“…Several reports have also suggested that the use of fusion partners such as GST [118], maltose-binding protein (MBP) [119,120], small ubiquitin-related modifier (SUMO) [87,121] and thioredoxin (Trx) [122] to the target proteins, especially to scFv fragments, results in solubility-enhancing properties and in increased yields of soluble and active products. However, the fusion partners must be cleaved as big tags usually interfere with the folding of the target protein and with its activity.…”

Section: Cytoplasmic Expressionmentioning

confidence: 99%

“…The SUMO fusion technology has been successfully applied to the cytosolic production of Fabs in E. coli. The highest yield of correctly folded and biologically active SUMO-tagged Fab, 12 mg/L, was recovered from cells harvested after a 16 h growth at 30 • C post-induction with 0.5 mM IPTG using the SHuffle strain and the EnBase medium [87].…”

Section: Cytoplasmic Expressionmentioning

confidence: 99%

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Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Sandomenico

Sivaccumar

Ruvo

2020

IJMS

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Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

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Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase^® cultivation mode

Abstract: The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.

Cited by 15 publications

References 40 publications

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

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Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode

Abstract: The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments.

Cited by 15 publications

References 40 publications

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

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Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase^® cultivation mode