Bacterial Inclusion Bodies: A Treasure Trove of Bioactive Proteins

Singhvi, Priyank; Saneja, Ankit; Srichandan, Sudeepa; Panda, Amulya K.

doi:10.1016/j.tibtech.2019.12.011

Cited by 97 publications

(65 citation statements)

References 89 publications

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“…Subsequently, before processing into various formats extensive dialysis is applied to remove excess denaturant, leading to extra dilution and precipitation. To overcome this phenomenon, recently, many mild [31,33,34]. Tag-free bioengineered spidroins were successfully prepared by thermal extraction and acidic extraction [27]; however, both methods are designed for soluble recombinant spidroins, while for spidroins in insoluble fraction high concentration denaturants are still necessary, which will break native-like secondary structures.…”

Section: Discussionmentioning

confidence: 99%

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Cai

Chen

et al. 2020

BMC Biotechnol

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Background: Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. Results: Here, we present a mild solubilization strategy-one-step heating method to solubilize spidroins from IBs, with combining spidroins' high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70-90°C for 20 min, pH 7.0-10, urea concentration 2-4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology. Conclusions: The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

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Section: Discussionmentioning

confidence: 99%

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Cai

Chen

et al. 2020

BMC Biotechnol

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show abstract

“…Subsequently, before processing into various formats extensive dialysis is applied to remove excess denaturant, leading to extra dilution and precipitation. To overcome this phenomenon, recently, many mild strategies for solubilizing IBs have been pursued for retaining native-like secondary structures, such as extreme pH, using detergents and freezing-thawing cycle [31,33,34]. Tag-free bioengineered spidroins were successfully prepared by thermal extraction and acidic extraction [27]; however, both methods are designed for soluble recombinant spidroins, while for spidroins in insoluble fraction high concentration denaturants are still necessary, which will break native-like secondary structures.…”

Section: Discussionmentioning

confidence: 99%

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Cheng¹,

Chen

Yu³

et al. 2020

Preprint

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Abstract Background: Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. Results: Here, we present a mild solubilization strategy—one-step heating method to solubilize spidroins from IBs, with combining spidroins’ high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70–90°C for 20 min, pH 7.0–10, urea concentration 2–4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology.Conclusions: The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

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“…During recombinant protein production, bacterial host cells split the overproduced heterologous proteins into soluble and insoluble fractions. The insoluble fraction is also known as inclusion bodies (IBs), which are biophysically described as protein nanomaterials with sizes ranging from 50 to 800 nm, and are easily formed during recombinant protein production [6][7][8]. During many years IBs have been considered a waste product [9].…”

Section: Introductionmentioning

confidence: 99%

Recombinant Protein-Based Nanoparticles: Elucidating Their Inflammatory Effects In Vivo and Their Potential as a New Therapeutic Format

et al. 2020

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Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties—intrinsic to IBs—with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.

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Bacterial Inclusion Bodies: A Treasure Trove of Bioactive Proteins

Cited by 97 publications

References 89 publications

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Recombinant Protein-Based Nanoparticles: Elucidating Their Inflammatory Effects In Vivo and Their Potential as a New Therapeutic Format

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