Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities.
The aim of the present study is to identify an effective and efficient expression system for purification of recombinant antiangiogenic protein tumstatin. The sequence encoding carboxy-terminal non-collagenous domain of α3 chain Type IV collagen, α3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLT-A to express it in bacteria, mammalian and Sf-9 insect cells respectively. Expression and purification profiles of tumstatin were evaluated by coomassie staining and immunoblotting, and the efficiency was determined based on the yields of soluble protein. Our results indicate that, baculovirus expression system was efficient for scalable yields of soluble protein that could be purified in its biologically active form. This baculovirus expressed tumstatin was used to evaluate its anti-angiogenic and anti-tumarogenic functions such as inhibition of endothelial cell proliferation, cell viability, migration, tube formation, cap dependent protein translation and the associated signaling mechanism including in-vivo tumor study. Our evaluated approaches using a modified baculovirus expression system shows high expression and high yield of biologically active tumstatin, as compared to two expression systems, indicating baculovirus expression system to be an ideal method for bulk production of soluble tumstatin that needed for pre-clinical and clinical trails.
Boosani
Chandra S
CS
Cell Signaling and Tumor Angiogenesis Laboratory, Department of Genetics, Boys Town National Research Hospital, Omaha, NE 68131, USA.
Varma
Ashok K
AK
Sudhakar
Akulapalli
A
Science. 2002 Jan 4;295(5552):140-3
11778052
J Biol Chem. 2000 Jul 14;275(28):21340-8
10766752
J Clin Invest. 1999 Jan;103(2):157-8
9916126
Gene Regul Syst Bio. 2007 Oct 14;1:217-26
19936090
J Biol Chem. 2000 Apr 14;275(15):10779-87
10753870
Nature. 1994 Oct 27;371(6500):762-7
7935836
J Biol Chem. 1993 Apr 25;268(12):8402-5
8473281
J Biol Chem. 2006 Jul 28;281(30):20932-9
16731529
Nat Rev Cancer. 2003 Jun;3(6):422-33
12778132
Invest Ophthalmol Vis Sci. 2009 Oct;50(10 ):4567-75
19443723
Blood. 2007 Aug 15;110(4):1168-77
17426256
Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4766-71
12682293
Am J Pathol. 2005 Mar;166(3):901-11
15743801
Cell. 1994 Oct 21;79(2):315-28
7525077
Nat Med. 2000 Dec;6(12 ):1335-40
11100117
J Biol Chem. 1994 Oct 14;269(41):25475-82
7929248
Cancer Res. 1999 Sep 15;59(18):4584-90
10493512
Nat Cell Biol. 2001 Jun;3(6):596-601
11389445
Cell. 1997 Jan 24;88(2):277-85
9008168
Protein Expr Purif. 2006 Oct;49(2):211-8
16631378
Circ Res. 2002 Jun 28;90(12):1243-50
12089061
Pharm Res. 2008 Dec;25(12):2731-9
18551250
FASEB J. 2006 Sep;20(11):1904-6
16877525
Exp Cell Res. 1997 Sep 15;235(2):403-12
9299165
J Biol Chem. 2004 Jan 23;279(4):2772-80
14610079
J Biol Chem. 1997 Aug 15;272(33):20395-401
9252346
J Clin Invest. 2005 Oct;115(10):2801-10
16151532
J Biol Chem. 1991 Aug 15;266(23):15318-24
1869555
Biochemistry. 1999 Nov 16;38(46):15398-405
10563826
Science. 1997 Jan 31;275(5300):628-30
9019819
Cell. 1992 Dec 11;71(6):891-4
1458538
Genes Dev. 2001 Apr 1;15(7):807-26
11297505
Curr Eye Res. 2010 Jan;35(1):45-55
20021254
Nat Med. 2002 Jan;8(1):27-34
11786903
Curr Opin Cell Biol. 1998 Apr;10(2):262-7
9561851
Matrix Biol. 2001 Sep;20(5-6):337-45
11566268
Proc Natl Acad Sci U S A. 1975 Apr;72(4):1441-5
1055416
J Biol Chem. 2000 Mar 17;275(11):8051-61
10713126
Biochemistry. 2000 Oct 24;39(42):12929-38
11041858