Expression of soluble, biologically active recombinant human tumstatin in Escherichia coli

Abstract

Tumstatin, a 28-kDa C-terminal fragment of collagen IV, is a potent anti-angiogenic protein and inhibitor of tumour growth. Recombinant tumstatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human tumstatin in E. coli by the coding region of tumstatin being linked to the 3'-end of the maltose-binding protein (MBP) gene. The fusion protein was expressed as the soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tumstatin was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 20% of the total soluble protein in E. coli, allowing approximately 8.6 mg of highly purified protein to be obtained per litre of bacterial culture. The purified tumstatin specifically inhibited the proliferation of endothelial cells in a dose-dependent manner. Annexin V-FITC apoptotic assay showed that recombinant tumstatin induced significant increase of apoptotic endothelial cells after 20 h of exposure to 20 microg/ml tumstatin, and when tumstatin was incubated on the chicken embryo, chorioallantoic membrane at doses of 1-15 microg, there was a dramatic decrease in the microvasculature allantoids of chicken embryos neovascular vessel test in vivo demonstrated that tumstatin treatment at doses of 1-15 microg gives rise to dramatically decrease the number of neovascular vessel. Our study provides a feasible and convenient approach to produce soluble and biologically active tumstatin.

Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM

“…The soluble form of the protein maintained the natural structure and function and it could be used for function analysis [ 35 ]. We purified the soluble protein from 4 L bacteria and 2 mg of purified APE was collected, a yield somewhat lower than previous reports [ 36 , 37 ]. Because the inclusion body was the main form of the immunotoxin, in follow-up work, we will purify the protein from inclusion bodies [ 38 ].…”

Cloning and expression of the tumstatin active peptides-T7 and its derivant-T7-NGR

“…An efficient expression system was introduced to us recently [11], it worked out with a MBP(maltose binding protein)-fusion vector. MBP-tumstatin was expressed and purified by amylose affinity chromatography.…”

Developments in purification methods for obtaining and evaluation of collagen derived endogenous angioinhibitors

“…Human recombinant tetrastatin [α4(IV)NC1] was also purified, recently by utilizing the His tags on Ni 2+ affinity columns, which inhibited melanoma cellular migration and in vivo tumor growth [39]. Fusion tag other than His tag also facilitated the purification of NC1 domains such as the maltose binding protein fusion tag was used for purification of soluble, anti-angiogenic, recombinant tumstatin from bacterial culture systems [40]. …”