NDLI: Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

Content Provider	Springer Nature : BioMed Central
Author	Hartinger, Doris Heinl, Stefan Schwartz, Heidi Elisabeth Grabherr, Reingard Schatzmayr, Gerd Haltrich, Dietmar Moll, Wulf-Dieter
Abstract	Background Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. Results When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG) used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3). Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3), which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the binding buffer were performed to obtain 1.45 mg of apparently homogeneous aminotransferase per liter of expression culture. Conclusions We found that only reduction of temperature, but not reduction of expression level or fusion to maltose-binding protein helped to produce correctly folded, active aminotransferase FumI in E. coli. Our results may provide a starting point for soluble expression of related aminotransferases or other aggregation-prone proteins in E. coli.
Related Links	https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/1475-2859-9-62.pdf
Ending Page	14
Page Count	14
Starting Page	1
File Format	HTM / HTML
ISSN	14752859
DOI	10.1186/1475-2859-9-62
Journal	Microbial Cell Factories
Issue Number	1
Volume Number	9
Language	English
Publisher	BioMed Central
Publisher Date	2010-08-18
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Binding Buffer Fusion Partner Immobilize Metal Affinity Chromatography Pyridoxal Phosphate Soluble Expression
Content Type	Text
Resource Type	Article
Subject	Applied Microbiology and Biotechnology Bioengineering Biotechnology
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.5/2023

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Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

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