Expression, purification and refolding of pro-MMP-2 from inclusion bodies of E. coli

Zhang, Yu Nan; Liu, Jia Jian; Zhang, Wei; Qin, Han; Wang, Lintao; Chen, Yuan Yuan; Li, Yuan; Yang, Fang; Cao, Rong; Wang, Xue Jun

doi:10.1016/j.pep.2023.106278

Cited by 2 publications

(4 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Full-length MMP-2 contains an autoinhibitory pro-domain, which must be removed (i.e., by proteolysis) for protease activation (Figure A). − More recently, the involvement of reactive oxygen/nitrogen species (RONS) has been shown in the cleavage-independent activation of MMP-2. ,, Full-length MMP-2 expressed in E. coli requires refolding after protein expression, and activation in vitro often by incubation with 4-aminophenylmercuric acetate (APMA) . We aimed to streamline the expression and purification process by improving the yield and stability of MMP-2.…”

Section: Resultsmentioning

confidence: 99%

“…Full-length MMP-2 can be expressed and purified from E. coli and mammalian cells. Expression in E. coli requires refolding following purification, 27,28 and expression of fulllength MMP-2 requires activation. 29 Our construct lacks the pro-domain and forgoes the requirement for additional refolding or activation steps.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Full-length MMP-2 can be expressed and purified from E. coli cells; however, this requires refolding and subsequent activation of the enzyme following purification. , Alternatively, expression in mammalian cells forgoes the requirement to refold MMP-2 after purification, an advantageous option . Both expression methods still require activation of MMP-2, often with 4-aminophenylmercuric acetate (APMA), which removes the inhibitory interaction of the pro-domain with the catalytic zinc, rendering MMP-2 active .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue

Hartley,

Bassiouni,

Roczkowsky

et al. 2024

J. Proteome Res.

View full text Add to dashboard Cite

Proteases are enzymes that induce irreversible posttranslational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue

Hartley,

Bassiouni,

Roczkowsky

et al. 2024

J. Proteome Res.

View full text Add to dashboard Cite

show abstract

“…MtbCsm2 was purified from inclusion bodies of E. coli . The purification process was carried out following established procedure ( Wang et al, 2009 ; Zhang et al, 2023 ), including protein inclusion body washing, inclusion body dissolving, urea gradient dialysis and protein refolding. The protein supernatant was pooled for further studies.…”

Section: Methodsmentioning

confidence: 99%

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis

Shi,

Zhang,

Fleming

et al. 2023

Front. Mol. Biosci.

View full text Add to dashboard Cite

Introduction:Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored.Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system.Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions.Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.

show abstract

Expression, purification and refolding of pro-MMP-2 from inclusion bodies of E. coli

Cited by 2 publications

References 31 publications

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis

Contact Info

Product

Resources

About