Prokaryotic Expression, Refolding and Purification of High-Purity Mouse Midkine in Escherichia coli

Gao, Jin; Wang, Haixia

doi:10.1007/s12010-015-1587-1

Cited by 3 publications

(2 citation statements)

References 16 publications

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“…Various methods have been developed to solubilize and renature the denatured proteins in inclusion bodies. Mouse midkine was expressed in E. coli as inclusion bodies, and the highly purified recombinant mouse midkine with bioactivity was generated following denaturing, refolding, ion exchange chromatography and affinity chromatography (Gao & Wang 2015). Palm tree peroxidases were highly expressed in E. coli as inclusion bodies, and their enzyme activities were shown to be restored after renaturation (Yuan et al 2021).…”

Section: Discussionmentioning

confidence: 99%

Prokaryotic expression and solubilisation of Arabidopsis ROOT UVB SENSITIVE 1 from inclusion bodies in Escherichia coli

Hou

Tong

2022

Bot Serb

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The RUS (ROOT UVB SENSITIVE 1) proteins characterized by their unique DUF647 domain are widely distributed in eukaryotes. Their functional roles are largely unknown except for the possible involvement of Arabidopsis RUS1 and RUS2 in early seedling development. To investigate the biochemical roles of the RUS proteins, full length and truncated Arabidopsis RUS1 were seamlessly fused with GFP and cloned into prokaryotic expression vector pQE-100 which allows proteins expressed with an N-terminal 6?His tag. Expression of the full length RUS1-GFP could not be detected after adding the inducer IPTG, while a truncated RUS1-GFP was expressed at high levels and formed inclusion bodies in Escherichia coli. The inclusion bodies were dissolved in a denaturing buffer, and then the truncated RUS1-GFP fusion protein in the supernatant was bound to a Ni-NTA slurry. The bound proteins were eluted after the non-specific binding proteins were washed away. The purified truncated proteins were detected as a single clear band of the expected size in SDS-PAGE, and were further confirmed by the Western blot test. Our results suggest that the impossible expression of the full length RUS1 protein in E. coli can be expressed in truncated form, and inclusion bodies can be effectively solubilized.

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Section: Discussionmentioning

confidence: 99%

Prokaryotic expression and solubilisation of Arabidopsis ROOT UVB SENSITIVE 1 from inclusion bodies in Escherichia coli

Hou

Tong

2022

Bot Serb

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“…43 The proteins were purified from inclusion bodies under denaturing conditions, and refolding was induced using a method described previously. 81, 82 To purify the 6xHis-tagged proteins, the harvested cells (150 ml of culture) were lysed in 3 ml of lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl [pH 8.0]) containing 6 M guanidinium chloride. The cell lysate was sonicated until clear, followed by centrifugation at 18,000xg for 30 min.…”

Section: Methodsmentioning

confidence: 99%

The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation

Sun

et al. 2016

Oncogene

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Flap endonuclease-1 (FEN1) is a multifunctional, structure-specific nuclease that plays a critical role in maintaining human genome stability. FEN1 mutations have been detected in human cancer specimens and have been suggested to cause genomic instability and cancer predisposition. However, the exact relationship between FEN1 deficiency and cancer susceptibility remains unclear. In the current work, we report a novel colorectal cancer-associated FEN1 mutation, L209P. This mutant protein lacks the FEN, exonuclease (EXO) and gap endonuclease (GEN) activities of FEN1 but retains DNA binding affinity. The L209P FEN1 variant interferes with the function of the WT FEN1 enzyme in a dominant negative manner and impairs long-patch base excision repair in vitro and in vivo. Expression of L209P FEN1 sensitizes cells to DNA damage, resulting in endogenous genomic instability and cellular transformation, as well as tumor growth in a mouse xenograft model. These data indicate that human cancer-associated genetic alterations in the FEN1 gene can contribute substantially to cancer development.

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Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System

Zhang

Sun

Dong

et al. 2017

Appl Biochem Biotechnol

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Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137-195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.

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Prokaryotic Expression, Refolding and Purification of High-Purity Mouse Midkine in Escherichia coli

Cited by 3 publications

References 16 publications

Prokaryotic expression and solubilisation of Arabidopsis ROOT UVB SENSITIVE 1 from inclusion bodies in Escherichia coli

Prokaryotic expression and solubilisation of Arabidopsis ROOT UVB SENSITIVE 1 from inclusion bodies in Escherichia coli

The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation

Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System

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