Refolding and Recovery of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) from Inclusion Bodies Expressed by Escherichia coli

Oneda, Hiroshi; Inouye, Kunlyo

doi:10.1093/oxfordjournals.jbchem.a022533

Cited by 60 publications

(44 citation statements)

References 9 publications

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“…After a 5-h induction, E. coli cells were broken in 50 mM Tris-HCl, pH 8.0 containing 50 mM NaCl and 5 mM EDTA with a sonicator, and the resultant inclusion bodies were collected by centrifugation. Solubilization of the inclusion bodies with guanidine-HCl followed by refolding by rapid dilution method was performed as described previously (35,36). In the case of GST fusion proteins, the refolded proteins were incubated with thrombin to remove the N-terminal GST region.…”

Section: Methodsmentioning

confidence: 99%

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

Higashi

Miyazaki

2008

Journal of Biological Chemistry

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When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor. Matrix metalloproteinases (MMPs)2 are a family of zincdependent endopeptidases that degrade components of the extracellular matrix (ECM) and are believed to play pivotal roles in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair, and tumor invasion (1, 2). The association of MMPs with cancer cell invasion and metastasis has suggested that these proteases represent attractive targets for the development of novel anti-tumor therapies. However, to date, no MMP inhibitor has been developed successfully as anti-tumor drugs mainly because of deleterious side effects. Recently, inhibition of some MMPs is considered to have protumorigenic effects (3). These effects may also partially account for the failure of the inhibitors in clinical trials. In both cases, the broad specificity of the MMP inhibitors must be a stiff obstacle for developing safe and effective drugs. Most MMPs are secreted as proMMP, an inactive zymogen that has an autoinhibitory propeptide in its N terminus and is proteolytically activated by serine proteases or some activated MMPs. The activities of activated MMPs are regulated by a family of inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). These physiological inhibitors also have broad specificity against MMPs; the activities of almost all MMPs are susceptible to TIMP (TIMP-1 to TIMP-4) inhibition, and some members of a disintegrin and a metalloproteinase family are also inhibited by these inhibitors (4 -6). To develop drugs for the treatment of diseases in which MMPs are involved, many hydroxamate-based inhibitors or other synthetic MMP inhibitors have been designed (7-10). Unfortunately, none of them is a specific inhibitor for individual MMPs. A common architecture of catalytic sites of MMPs probably relates to the broad specificity of the inhibitors.Among the MMP family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are critical in the invasion of tumor cells across basement membranes, because of their strong activity against type IV collagen, a major component of basement membranes (11-13). Although they have a common substrate, MMP-2 is categorized as a good target for antican-* This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental data, Fig. S1, and Tables SI and SII

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Section: Methodsmentioning

confidence: 99%

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

Higashi

Miyazaki

2008

Journal of Biological Chemistry

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“…Expression and Purification of Recombinant Matrilysin-Expression of glutathione S-transferase-pro-matrilysin-FLAG, glutathione S-transferase-pro-E215A-FLAG, and solubilization of inclusion bodies with guanidine-HCl followed by refolding were performed as described previously (20,21). To remove the N-terminal glutathione S-transferase region, the refolded proteins were incubated with thrombin, and resultant pro-matrilysin and pro-E215A were purified, using an anti-FLAG M2 monoclonal antibody-conjugated agarose column.…”

Section: Methodsmentioning

confidence: 99%

Binding of Active Matrilysin to Cell Surface Cholesterol Sulfate Is Essential for Its Membrane-associated Proteolytic Action and Induction of Homotypic Cell Adhesion

Yamamoto

Higashi

Kioi

et al. 2006

Journal of Biological Chemistry

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Regulation of cell surface molecules by matrix metalloproteinases (MMPs), as well as MMPs-catalyzed degradation of extracellular matrix, is important for tumor invasion and metastasis. Our previous study (Kioi, M., Yamamoto, K., Higashi, S., Koshikawa, N., Fujita, K., and Miyazaki, K. (2003) Oncogene 22, 8662-8670) demonstrated that active matrilysin specifically binds to the surface of colon cancer cells and induces notable cell aggregation due to processing of the cell membrane protein(s). Furthermore, these aggregated cells showed a dramatically enhanced metastatic potential. To elucidate the mechanism of matrilysin-induced cell aggregation, we attempted to identify the matrilysin-binding substance on the cell surface. Here, we demonstrate that cholesterol sulfate on the cell surface is a major matrilysin-binding substance. We found that active matrilysin bound to the cell membrane and cholesterol sulfate incorporated into liposomes with similar affinities. Treatment of colon cancer cells with ␤-cyclodextrin significantly reduced not only matrilysin binding to the cell surface but also matrilysin-dependent proteolysis and cell aggregation. Interestingly, replenishment of cholesterol sulfate, but not cholesterol, neutralized the effects of ␤-cyclodextrin. Taken together, it is likely that binding of matrilysin to cholesterol sulfate facilitates the matrilysin-catalyzed modulation of cell surface proteins, thus inducing the cancer cell aggregation. Matrix metalloproteinases (MMPs)2 are zinc-dependent endopeptidases that degrade components of the extracellular matrix and play essential roles in tissue remodeling in physiological and pathological processes such as morphogenesis, differentiation, angiogenesis, tissue remodeling, and tumor invasion (1, 2). The association of MMPs with cancer cell invasion and metastasis has suggested that these proteinases represent attractive targets for the development of novel anti-tumor therapies.Recently, it has been suggested that the biological functions of various cell surface proteins are proteolytically modulated by several MMPs (1, 3, 4), including MT-MMPs, gelatinase A (MMP-2), gelatinase B (MMP-9), stromelysin (MMP-3), and matrilysin (MMP-7). These metalloproteinases are thought to regulate cellular functions by activating, inactivating, or releasing membrane proteins. Such regulation of cell surface molecules as well as MMP-catalyzed degradation of the extracellular matrix is important for tumor invasion and metastasis.Matrilysin is the smallest member of the MMP family, which lacks the C-terminal hemopexin-like domain. In cancer tissues, various MMPs are overexpressed both in stromal and tumor cells. In contrast, matrilysin has been detected specifically in tumor cells but not in stromal cells (5, 6). Among many MMPs, matrilysin appears to be one of the most important MMPs in human colon cancers because it is almost always overexpressed in colon cancers. Expression of matrilysin is also correlated well with malignancy and metastasis of cancers, especially in liver me...

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“…ARGININE was first used in refolding of human tissue type plasminogen activator [18,19]. Since then, it has been used for refolding of a variety of proteins including casein kinase II, [20], Fab antibody fragments [18,21], growth hormone [22], human gamma interferon, [23], human matrix metalloproteinase-7 human neurotrophins [24] human p53 tumor suppressor protein [25], interleukin-21 [26], interleukin-6 receptor [27], lysozyme [28], single-chain Fv fragments [29], and singlechain immunotoxins [18,30]. However, arginine may act as a protein-denaturant, which limits the expansion of its applications.…”

Section: Arginine (Arg)mentioning

confidence: 99%

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Alibolandi¹,

Mirzahosei²

2011

American J. of Biochemistry and Molecular Biology

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Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

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Refolding and Recovery of Recombinant Human Matrix Metalloproteinase 7 (Matrilysin) from Inclusion Bodies Expressed by Escherichia coli

Cited by 60 publications

References 9 publications

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

Binding of Active Matrilysin to Cell Surface Cholesterol Sulfate Is Essential for Its Membrane-associated Proteolytic Action and Induction of Homotypic Cell Adhesion

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

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