Journal: Biochemistry

Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

doi: 10.1021/acs.biochem.7b00700

Figure Lengend Snippet: Comparison of wild-type (wt) TIMP-2 (pT2MO1 coding sequence) and codon-optimized (CO) rhTIMP-2-6XHis (COT2his) cDNA and protein sequences. (A) ClustalW (MacVector version 15.5.0) pairwise nucleotide alignment and comparison of the wt TIMP-2 (pT2MO1) and codon-optimized rhTIMP-2-6XHis (COT2his) cDNA sequences using a 10-nucleotide window indicating changes made to and from G and C nucleotides. (B) Comparison of the % GC content in the pT2MO1 (blue) and COT2his (red) lines in 10-nucleotide sequence windows using the MacVector software. (C) Alignment of the translated protein sequences from wt (pT2MO1) and codon-optmized rhTIMP-2-6XHis (COT2his) cDNA sequences.

Article Snippet: Separated proteins were blotted and probed with the murine anti-TIMP-2 antibody (catalog no. MAB 9711, R&D Systems) and anti-His-6 (catalog no. MA1–21315, Pierce).

Techniques: Sequencing, Software

Journal: Biochemistry

Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

doi: 10.1021/acs.biochem.7b00700

Figure Lengend Snippet: Analysis of two-step downstream purification of rhTIMP-2– 6XHis following bioprocess scale expression. Samples from the bioprocess purification were analyzed as shown in the (A) PageBlue Protein-stained SDS−PAGE gel of the fractions obtained from the IMAC (HisTrap column) purification step. Lane M contained the molecular weight standards (SeeBlue Plus 2 Prestained Standards, Invitrogen, catalog no. LC 5925). Lane CM contained the starting condition medium sample obtained from the HEK-293-F suspension culture. Lane FT contained the flow-through (unbound) fraction obtained during sample loading. Lane NSE contained the nonspecific elution obtained during step gradient elution with 20 mM imidazole. Lane SE contained the specific elution fraction obtained following 250 mM imidazole step elution. (B) PageBlue stained SDS−PAGE gel showing that the rhTIMP-2-6XHis-containing fractions from the IMAC (HisTrap) purification contain a predominant 22 kDa band with minor higher-molecular weight contaminants. The reverse phase HPLC purification effectively removed these higher-molecular weight contaminants, resulting in a single 22 kDa band with >95% purity as estimated by densitometry using a Bio-Rad ChemiDoc XRS+ instrument with Image Lab software. (C) Western Blot analysis of the IMAC fractions using anti-TIMP-2 (top) anti-6XHis tag (bottom) antibodies. The lanes are labeled the same as in panel A.

Article Snippet: Separated proteins were blotted and probed with the murine anti-TIMP-2 antibody (catalog no. MAB 9711, R&D Systems) and anti-His-6 (catalog no. MA1–21315, Pierce).

Techniques: Purification, Expressing, Staining, SDS Page, Molecular Weight, Software, Western Blot, Labeling

Journal: Biochemistry

Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

doi: 10.1021/acs.biochem.7b00700

Figure Lengend Snippet: Inhibition of MMP-2 activity by rhTIMP-2-6XHis and Ala +TIMP-2 was monitored by colorimetric product formation at 412 nm. (A) MMP-2 enzymatic activity data plotted vs log molar concentration of rhTIMP-2-6XHis (red) and Ala+TIMP-2 (blue) were fitted to a nonlinear curve for the one-site enzyme inhibitor response. (B) Data from the linear range of the rhTIMP-2-6XHis inhibition curve were analyzed by linear regression analysis and extrapolation to the X-axis to estimate the molar ratio of rhTIMP-2-6XHis to MMP-2, resulting in complete inhibition of MMP-2 enzymatic activity (n = 3; mean ± standard error of the mean).

Article Snippet: Separated proteins were blotted and probed with the murine anti-TIMP-2 antibody (catalog no. MAB 9711, R&D Systems) and anti-His-6 (catalog no. MA1–21315, Pierce).

Techniques: Inhibition, Activity Assay, Concentration Assay

Journal: The Journal of International Medical Research

Article Title: Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury

doi: 10.1177/0300060520919592

Figure Lengend Snippet: Primer sequences for real-time quantitative polymerase chain reaction analyses.

Article Snippet: Mouse anti-rat MMP9 (cat. no. NBP2-13173SS) and TIMP2 antibodies (cat. no. NBP1-42375) were purchased from Novus Biologicals Inc. (Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: The Journal of International Medical Research

Article Title: Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury

doi: 10.1177/0300060520919592

Figure Lengend Snippet: Lung MMP and TIMP mRNA and protein expression. Changes in MMP2 (a), TIMP2 (b), MMP2/TIMP2 (c), MMP9 (d), TIMP1 (e), and MMP9/TIMP1 (f) in all groups treated with or without LPS. (g) Protein expression in each group as detected by western blotting. LPS 2h, 6h, 12h, and 24h indicate 2, 6, 12, and 24 hours after LPS injection in the acute lung injury group, respectively. Data are shown as mean ± standard deviation (n = 6 per group). * P < 0.05 vs. control, # P < 0.05 vs. LPS 2h. Control, normal control group; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; LPS, lipopolysaccharide

Article Snippet: Mouse anti-rat MMP9 (cat. no. NBP2-13173SS) and TIMP2 antibodies (cat. no. NBP1-42375) were purchased from Novus Biologicals Inc. (Littleton, CO, USA).

Techniques: Expressing, Western Blot, Injection, Standard Deviation, Control

Journal: Disease markers

Article Title: Combination of Strong MMP-2 and Weak TIMP-2 Immunostainings Is a Significant Prognostic Factor in Endometrial Carcinoma

doi: 10.1155/2013/416870

Figure Lengend Snippet: The independent and significant prognostic factors for the cancer-specific survival of endometrial cancer patients as evaluated by the Cox regression method. Relative risk of death, its 95% confidence interval, and P values are given for each covariate. n = 225.

Article Snippet: A mouse monoclonal antibody to MMP-2 (CA-4001, Diabor Ltd., Oulu, Finland) or TIMP-2 (MAB 971, R&D Systems, Minneapolis, MN, USA) was used as the primary antibody.

Techniques: