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One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies.
BMC Biotechnol. 2020 Jul 10; 20(1):37.BB

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.

Authors+Show Affiliations

The Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, Jiangsu, China.Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, 14157, Huddinge, Sweden.The Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, Jiangsu, China.The Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, Jiangsu, China.The Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, Jiangsu, China. sdxiongfd@126.com.The Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, Jiangsu, China. xmqi@suda.edu.cn.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32650749

Citation

Cai, Hui, et al. "One-step Heating Strategy for Efficient Solubilization of Recombinant Spider Silk Protein From Inclusion Bodies." BMC Biotechnology, vol. 20, no. 1, 2020, p. 37.
Cai H, Chen G, Yu H, et al. One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies. BMC Biotechnol. 2020;20(1):37.
Cai, H., Chen, G., Yu, H., Tang, Y., Xiong, S., & Qi, X. (2020). One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies. BMC Biotechnology, 20(1), 37. https://doi.org/10.1186/s12896-020-00630-1
Cai H, et al. One-step Heating Strategy for Efficient Solubilization of Recombinant Spider Silk Protein From Inclusion Bodies. BMC Biotechnol. 2020 Jul 10;20(1):37. PubMed PMID: 32650749.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies. AU - Cai,Hui, AU - Chen,Gefei, AU - Yu,Hairui, AU - Tang,Ying, AU - Xiong,Sidong, AU - Qi,Xingmei, Y1 - 2020/07/10/ PY - 2020/03/12/received PY - 2020/07/02/accepted PY - 2020/7/12/entrez PY - 2020/7/12/pubmed PY - 2020/7/12/medline KW - Heating KW - Inclusion body solubilization KW - NM IBs KW - Recombinant spidroin SP - 37 EP - 37 JF - BMC biotechnology JO - BMC Biotechnol. VL - 20 IS - 1 N2 - 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. SN - 1472-6750 UR - https://www.unboundmedicine.com/medline/citation/32650749/One-step_heating_strategy_for_efficient_solubilization_of_recombinant_spider_silk_protein_from_inclusion_bodies L2 - https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-020-00630-1 DB - PRIME DP - Unbound Medicine ER -
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