Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity

Fangfang Fan1 Huiying Sun1 Hui Xu1 Jiawei Liu1 Haiyuan Zhang1 Yilan Li1 Xuelian Ning1 Yue Sun1 Jing Bai1 Songbin Fu1 Chunshui Zhou1

(1.Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang, China 150081)

【Abstract】AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing Att B sites were designed and used to create the Att B-TEV-FLAG-AIK fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by att B and att P mediated recombination (BP reaction), then, transferred into the destination vector pDEST15 by att L and att R mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 E. coli transformed by the GST-AIK expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIK fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIK on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 E. coli with starting OD600 at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.

【Keywords】 cationic peptide; site-specific recombination; GST fusion protein; inducible expression; affinity purification; antitumor activity;

【DOI】

【Funds】 National Natural Science Foundation of China (No. 81272582) National Natural Science Foundation of China(No.81272582)

Download this article

    References

    [1]Zhang R, Lao XZ, Zheng H. Research progress on small-molecule antitumor peptides. Amino Acids Biotic Resour, 2012, 34(4):42–46 (In Chinese).

    [2]Zhao R, Meng QY, Deng X, et al. Research progress on peptides as antitumor agent. Strait Pharmac J, 2012, 24(10):4–8 (In Chinese).

    [3]Oyston PCF, Fox MA, Richards SJ, et al. Novel peptide therapeutics for treatment of infections. JMed Microbiol, 2009, 58 (Pt 8):977-987.

    [4]Wu DD, Gao YF, Qi YM, et al. Peptide-based cancer therapy: opportunity and challenge. Cancer Lett, 2014, 351(1):13–22.

    [5]Alberici L, Roth L, Sugahara KN, et al. De novo design of a tumor-penetrating peptide. Cancer Res, 2013, 73(2):804–812.

    [6]Zahid M, Lu X, Mi Z, et al. 4-Cationic and tissue-specific protein transduction domains:identification, characterization, and therapeutic application. Adv Genet, 2010, 69:83–95.

    [7]Fan FF, Xu H, Sun HY, et al. Antitumor activity of a novel synthetic cationic peptide AIK: a preliminary observation. Chin J Cancer Biother, 2014, 21(6):617–623 (In Chinese).

    [8]Lee JY, Kang SK, Li HS, et al. Production of recombinant human growth hormone conjugated with a transcytotic peptide in Pichia pastoris for effective oral protein delivery. Mol Biotechnol, 2015, 57(5):430–438.

    [9]Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol, 2014, 5:172.

    [10]Schägger H. Tricine-SDS-PAGE. Nat Protoc, 2006, 1(1):16–22.

    [11]Berge G, Eliassen LT, Camilio KA, et al. Therapeutic vaccination against a murine lymphoma by intratumoral injection of a cationic anticancer peptide. Cancer Immunol Immunother, 2010, 59(8):1285–1294.

    [12]Mandal SM, Migliolo L, Das S, et al. Identification and characterization of a bactericidal and proapoptotic peptide from Cycas revoluta seeds with DNA binding properties. J Cell Biochem, 2012, 113(1):184–193.

    [13]Martin-Algarra S, Espinosa E, RubióJ, et al. Phase Ⅱ study of weekly Kahalalide F in patients with advanced malignant melanoma. Eur Cancer, 2009, 45(5):732–735.

    [14]Yang WH, Luo DF, Wang SX, et al. TMTP1, a novel tumor-homing peptide specifically targeting metastasis. Clin Cancer Res, 2008, 14(17):5494–5502.

    [15]Riely GJ, Gadgeel S, Rothman I, et al. A phase 2study of TZT-1027, administered weekly to patients with advanced non-small cell lung cancer following treatment with platinum-based chemotherapy. Lung Cancer, 2007, 55(2):181–185.

    [16]Hartley JL, Temple GF, Brasch MA. DNA cloning using in vitro site-specific recombination. Genome Res, 2000, 10(11):1788–1795.

    [17]Lamesch P, Li N, Milstein S, et al. h ORFeome v3. 1:a resource of human open reading frames representing over 10 000 human genes. Genomics, 2007, 89(3):307–315.

    [18]Emanuele MJH, Elia AE, Xu QK, et al. Global identification of modular cullin-RING ligase substrates. Cell, 2011, 147(2):459–474.

    [19]Walhout AJM, Temple GF, Brasch MA, et al. GATEWAY recombinational cloning:application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol, 2000, 328:575–592.

    [20]Arabidopsis interactome mapping consortium evidence for network evolution in an Arabidopsis interactome map. Science, 2011, 333(6042):601–607.

    [21]Yu H, Ma Q, Lin J, et al. Expression and purification of GST-FHL2 fusion protein. Genet Mol Res, 2013, 12(4):6372–6378.

    [22]Rabhi-Essafi I, Sadok A, Khalaf N, et al. A strategy for high-level expression of soluble and functional human interferonαas a GST-fusion protein in E. coli. Protein Eng Des Sel, 2007, 20(5):201–209.

    [23]Gupta A, Rath PC. Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L. J Biosci, 2012, 37(1):103–113.

    [24]Pandey M, Rath PC. Expression of interferon-inducible recombinant human RNase Lcauses RNA degradation and inhibition of cell growth in Escherichia coli. Biochem Biophys Res Commun, 2004, 317(2):586–597.

    [25]Tang WH, Zhang JL, Wang ZY, et al. The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE. Acta Phytophysiol Sin, 2000, 26(1):64–68 (In Chinese).

This Article

ISSN:1000-3061

CN:11-1998/Q

Vol 31, No. 12, Pages 1753-1763

December 2015

Downloads:0

Share
Article Outline

Knowledge

Abstract

  • 1 Materials and methods
  • 2 Results
  • 3 Discussion
  • References