Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human α1 type IV collagen expressed in Sf9 cells

doi:10.1016/j.pep.2006.03.007

Protein Expression and Purification

Volume 49, Issue 2, October 2006, Pages 211-218

https://doi.org/10.1016/j.pep.2006.03.007 Get rights and content

Abstract

α1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human α1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of α1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize α1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of α1(IV)NC1 is needed. In the present study, expression of α1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.

Section snippets

Materials and methods

Primary human umbilical vein endothelial cells (HUVECs) were purchased from Clonetech^™. Recombinant human bFGF was obtained from R&D systems, Inc. Horseradish peroxidase (HRP)-labeled secondary antibodies and penicillin/streptomycin were purchased from Sigma–Aldrich. BD Matrigel^™ Matrix (14.6 mg/ml) was purchased from BD Biosciences Discovery Laboratory. FBS, ECL Kit was obtained from Amersham biosciences; Graces insect cell culture medium, cell fixer, and staining H&E were purchased form Fisher

Cloning of α1(IV)NC1 in baculovirus transfer vector pAcHLT-A

Total RNA from human kidney was isolated using TriZol Reagent. The cDNA encoding active domain of α1 type IV NC1 (690 bp) was PCR amplified using SuperScript OneStep RT-PCR system with platinum Taq and primer sequences described under Materials and methods (Fig. 1A). The recombinant clones were sequenced using high-throughput DNA sequencing facility at University of Nebraska Medical Centre. The sequences were further confirmed by BLAST search. The structure of α1(IV)NC1 was diagrammatically

Acknowledgments

This study was supported by Dobelman Head and Neck Cancer grant research funds of Cell Signaling and Angiogenesis Laboratory, Usher Center at Boys Town National Research Hospital to Sudhakar Akulapalli. We thank Dr. Barbara J. Morley for her help with inverted microscope.

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Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human α1 type IV collagen expressed in Sf9 cells

Abstract

Section snippets

Materials and methods

Cloning of α1(IV)NC1 in baculovirus transfer vector pAcHLT-A

Acknowledgments

Cell

J. Biol. Chem.

Kidney Int.

J. Biol. Chem.

Matrix Biol.

Am. J. Pathol.

J. Biol. Chem.

Biochem. Pharmacol.

Tumor angiogenesis: therapeutic implications

N. Engl. J. Med.

Inhibition of angiogenesis through modulation of collagen metabolism

Lab. Invest.