Expression, Purification, and Characterization of Recombinant Protein GX1-rmhTNFα

Abstract

A phage-displayed peptide CGNSNPKSC (GX1) was obtained previously in our lab, which could specifically bind to the vasculature of human gastric cancer. GX1-rmhTNFalpha was a fusion protein of GX1 and recombinant mutant human tumor necrosis factor alpha (rmhTNFalpha), which was designed by us with the expectation of enhancing selectivity of rmhTNFalpha. The DNA fragment encoding GX1 was cloned into the vector pBV220 with rmhTNFalpha between the EcoRI site and the BamHI site, and then expressed in Escherichia coli DH5alpha by temperature induction. Subsequently, E. coli DH5alpha was lysed, and the GX1-rmhTNFalpha protein was found in both soluble form and inclusion bodies. The protein was fractionated with ammonium sulfate deposition from 30% to 60%, and purified by cation and anion exchange chromatography using SP Sepharose Fast Flow column and Q Sepharose Fast Flow column. The purity of protein was then identified by SDS-PAGE and HPLC. Subsequent studies showed that GX1-rmhTNFalpha had high bioactivity of 5.65 x 10(8) IU/ml, which was similar with natural human TNFalpha and could reach the tumor site relatively faster than rmhTNFalpha.

In vivo quantifying molecular specificity of Cy55-labeled cyclic 9-mer peptide probe with dynamic fluorescence imaging

“…GX1 (CGNSNPKSC), a cyclic 9-mer peptide, was identified by Min Zhi et al in 2004 by using in vivo phage display technology [1], which binds specifically to the gastric tumor-derived vascular endothelial cells. Further research confirmed that GX1 was capable of effective specific targeting of the tumor vasculature and could be applied to anti-angiogenesis iatreusis of cancer, together with anti-neoplastic agents, as a novel tumor vascular marker [2][3][4][5][6]. At the same time, GX1 was successfully conjugated with a fluorescing cyanine dye, Cy5.5, for optical imaging in vivo by Kai Chen et al in 2012 [7].…”

“…Animal Care and all experiments were performed in accordance with the internationally recognized guidelines. About 200 ml BGC-823 cancer cells suspended in PBS (1*10 7 cells/ml) were implanted subcutaneously into the right shoulder of nude mice and grown for about 14 days when the tumor reached approximately 300 mm 3 [6]. Twelve mice were randomly divided into three groups.…”

Investigation of injection dose and camera integration time on quantifying pharmacokinetics of a Cy5.5-GX1 probe with dynamic fluorescence imagingin vivo

“…5 Correspondingly, a cyclic 9-mer peptide named GX1 was identified by Zhi et al, 6 which exhibits the specific ability to target vasculature of gastric cancer. [7][8][9] Furthermore, the GX1 peptide was successfully conjugated with near-infrared (NIR) fluorescent dye Cy5.5 to obtain the Cy5.5-GX1 probe, which could be used for tumor-targeted imaging and detection. 10 Unfortunately, the in vivo pharmacokinetic properties of GX1 have not been fully studied yet.…”

Evaluation of Tc-99 m Labeled Dimeric GX1 Peptides for Imaging of Colorectal Cancer Vasculature

“…GX1 was identified by Min Zhi et al in 2004 by screening a phage-displayed peptide library in vivo [5] and was appraised and further studied by Xiaoli Hui et al [6][7][8][9][10]. Previous results indicated that GX1 was able to bind specifically to the tumor vasculature and could be applied to anti-angiogenesis iatreusis of cancer, together with anti-neoplastic agents, as a novel tumor vascular marker.…”