Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator
被引:17
作者:
论文数: 引用数:
h-index:
机构:
Kaczmarek, Jakub Zbigniew
[1
]
Skottrup, Peter Durand
论文数: 0引用数: 0
h-index: 0
机构:
Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, DK-2650 Hvidovre, DenmarkUniv Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, DK-2650 Hvidovre, Denmark
Skottrup, Peter Durand
[1
]
机构:
[1] Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, DK-2650 Hvidovre, Denmark
Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumouts and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naive library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naive library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure-function relationships. (C) 2015 Elsevier Ltd. All rights reserved.
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Ass, Kathleen
;
Ahmad, Aamir
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Ahmad, Aamir
;
Azmi, Asfar S.
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Azmi, Asfar S.
;
Sarkar, Sarah H.
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Sarkar, Sarah H.
;
Sarkar, Fazlul H.
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Ass, Kathleen
;
Ahmad, Aamir
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Ahmad, Aamir
;
Azmi, Asfar S.
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Azmi, Asfar S.
;
Sarkar, Sarah H.
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA
Sarkar, Sarah H.
;
Sarkar, Fazlul H.
论文数: 0引用数: 0
h-index: 0
机构:
Wayne State Univ, Sch Med, Detroit, MI 48201 USA
Barbara Ann Karmanos Canc Ctr, Dept Pathol, Detroit, MI 48201 USAWayne State Univ, Sch Med, Detroit, MI 48201 USA