Expression of cloned monkey metallothionein in Escherichia coli

Murooka, Yoshikatsu; Nagaoka, Tetsuya

doi:10.1128/aem.53.1.204-207.1987

Cited by 40 publications

(5 citation statements)

References 14 publications

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“…Earlier attempts to produce MTs in bacterial cells (i.e., Escherichia coli) as a way to increase their metal-binding ability were successful in some cases (2,22,31,37). However, expression of such Cys-rich proteins is not devoid of problems because of the predicted interference with the redox pathways in the cytosol (1,30,34,35,45).…”

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confidence: 99%

Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

et al. 1998

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Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153-YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. colicells to bind Cd2+ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.

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confidence: 99%

Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

et al. 1998

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“…Expression and localization within the periplasm. The expression of small proteins in E. coli has often been problematic due to instability and degradation (18,21). This is especially true in cases where the protein is repetitive in nature.…”

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“…This is especially true in cases where the protein is repetitive in nature. These problems have been minimized by expressing these proteins as fusion proteins (18,21,25,26). We previously expressed the N. crassa metallothionein gene within the cytoplasm and periplasm of E. coli by using the fusion protein of vector pMAL-c and pMAL-p (encoding the maltose binding protein), respectively (21).…”

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confidence: 99%

Development of Bacterium-Based Heavy Metal Biosorbents: Enhanced Uptake of Cadmium and Mercury by Escherichia coli Expressing a Metal Binding Motif

Pazirandeh

Wells

Ryan

1998

Appl Environ Microbiol

124

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A gene coding for a de novo peptide sequence containing a metal binding motif was chemically synthesized and expressed inEscherichia coli as a fusion with the maltose binding protein. Bacterial cells expressing the metal binding peptide fusion demonstrated enhanced binding of Cd2+ and Hg2+compared to bacterial cells lacking the metal binding peptide. The potential use of genetically engineered bacteria as biosorbents for the removal of heavy metals from wastewaters is discussed.

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“…Although metallothioneins were for many years purified from animal tissues, mostly the liver in the case of mammals, currently the most common technique for obtaining particular homogenous MT isoforms is the production of recombinant protein directly or in the form of a fusion protein from E. coli [ 17 – 20 ]. However, the low stability and cell toxicity of high thiol content in MT hinders its high expression in E. coli [ 21 – 25 ]. Various fusion tags such as ubiquitin (Ub), glutathione S-transferase (GST), S-tag, maltose binding protein (MBP), etc., have been shown to enhance protein stability by acting as chaperones, and frequently the fusion protein is expressed as a soluble protein rather than in inclusion bodies.…”

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confidence: 99%

Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3

Singh

Krężel

2023

Microb Cell Fact

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Background Mammalian metallothioneins (MTs) are small (6–7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies. Results Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recognition motif (LPETG) as removable fusion tags for high-level expression and purification of human MT3 from the bacterial system. In the first strategy, SUMOylated MT3 was expressed and purified using Ulp1-mediated cleavage. In the second strategy, SUMOylated MT3 with a sortase recognition motif at the N-terminus of MT3 was expressed and purified using sortase-mediated cleavage. In the final strategy, the fusion protein His6-SUMO-eSrtA-LPETG-MT3 was expressed and purified by one-step sortase-mediated inducible on-bead autocleavage. Using these three strategies the apo-MT3 was purified in a yield of 11.5, 11, and 10.8 mg/L, respectively, which is the highest yield achieved for MT expression and purification to date. No effect of MT3 on Ni2+-containing resin was observed. Conclusion The SUMO/sortase-based strategy used as the production system for MT3 resulted in a very high expression level and protein production yield. The apo-MT3 purified by this strategy contained an additional glycine residue and had similar metal binding properties as WT-MT3. This SUMO-sortase fusion system is a simple, robust, and inexpensive one-step purification approach for various MTs as well as other toxic proteins with very high yield via immobilized metal affinity chromatography (IMAC).

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Expression of cloned monkey metallothionein in Escherichia coli

Cited by 40 publications

References 14 publications

Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

Development of Bacterium-Based Heavy Metal Biosorbents: Enhanced Uptake of Cadmium and Mercury by Escherichia coli Expressing a Metal Binding Motif

Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3

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