Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF
Granulocyte colony-stimulating factor (G-CSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of G-CSF has been implicated in several important processes in tumor biology such as tumor growth, angiogenesis, and metastasis. Four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF (transcript variants 1, 2, 3 and 4). The mRNAs and protein products of splice variants 1 and 2 have been isolated for the first time, from tumor cell lines. In the present study for the first time we isolated the G-CSF transcript variant 4 encoding G-CSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of G-CSF. Both the full-length G-CSF isoform B and G-CSF isoform D were cloned from Mehr80 cell line, overexpressed in Escherichia coli as N-terminal glutathione-S-transferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathione-Sepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X-114 and CHAPS and purified. The purity of G-CSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and they were clearly detected in western blot analysis using anti-G-CSF polyclonal antibody. The G-CSF plays various roles in physiological and pathological conditions, however to date, the differential function of G-CSF isoforms remains unknown. Considering the fact that G-CSF isoform D was isolated from a highly malignant tumor cell line with overexpression of G-CSF, the role of this splice variant in tumorigenesis requires further investigation.
2. Dale DC, Bonilla MA, Davis MW, Nakanishi AM, et al. A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia. Blood 1993; 81(10):2496-502.
3. Nagata S, Tsuchiya M, Asano S, Yamamoto O, Hirata Y, Kubota N, et al. The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO J 1986; 5(3):575-81.
4. Chung AS, Wu X, Zhuang G, Ngu H, Kasman I, Zhang J, et al. An interleukin-17–mediated paracrine network promotes tumor resistance to anti-angiogenic therapy. Nat Med 2013; 19(9):1114-23.
5. Kowanetz M, Wu X, Lee J, Tan M, et al. Granulocyte-colony stimulating factor promotes lung metastasis through mobilization of Ly6G+Ly6C+ granulocytes. Proc Natl Acad Sci U S A 2010; 107(50):21248-55.
6. Shojaei F, Wu X, Qu X, Kowanetz M, Yu L, Tan M, et al. G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models. Proc Natl Acad Sci U S A 2009; 106(16):6742-7.
7. Waight JD, Hu Q, Miller A, Liu S, Abrams SI. Tumor-Derived G-CSF Facilitates Neoplastic Growth through a Granulocytic Myeloid-Derived Suppressor Cell-Dependent Mechanism. PLoS ONE 2011; 6(11):e27690.
8. Granger JM, Kontoyiannis DP. Etiology and outcome of extreme leukocytosis in 758 nonhematologic cancer patients: a retrospective, single-institution study. Cancer 2009; 115(17):3919-23.
9. Demetri GD, Griffin JD. Granulocyte colony-stimulating factor and its receptor. Blood 1991; 78(11):2791-808.
10. Kawano M, Mabuchi S, Matsumoto Y, Sasano T, Takahashi R, Kuroda H, et al. The significance of G-CSF expression and myeloid-derived suppressor cells in the chemoresistance of uterine cervical cancer. Sci Rep 2015; 5:18217.
11. Chakraborty A, Guha S. Granulocyte colony-stimulating factor/granulocyte colony-stimulating factor receptor biological axis promotes survival and growth of bladder cancer cells. Urology 2007; 69(6):1210-5.
12. Beekman R, Touw IP. G-CSF and its receptor in myeloid malignancy. Blood 2010; 115(25):5131-6.
13. Ehlers S, Herbst C, Zimmermann M, Scharn N, Germeshausen M, von Neuhoff N, et al. Granulocyte colony-stimulating factor (G-CSF) treatment of childhood acute myeloid leukemias that overexpress the differentiation-defective G-CSF receptor isoform IV is associated with a higher incidence of relapse. J Clin Oncol 2010; 28(15):2591-7.
14. Nomura H, Imazeki I, Oheda M, Kubota N, Tamura M, Ono M, et al. Purification and characterization of human granulocyte colony-stimulating factor (G-CSF). EMBO J 1986; 5(5):871-6.
15. Welte K, Platzer E, Lu L, Gabrilove JL, Levi E, Mertelsmann R, et al. Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc Natl Acad Sci U S A 1985; 82(5):1526-30.
16. Sharifzadeh S, Owji SM, Pezeshki AM, Malek-Hoseini Z, Kumar PV, Ghayumi SM, et al. Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation. Pathol Oncol Res 2004; 10(4):225-30.
17. Oltean S, Bates DO. Hallmarks of alternative splicing in cancer. Oncogene 2014; 33(46):5311-8.
18. Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques 2010; 48(1):61-4.
19. Francis DM, Page R. Strategies to Optimize Protein Expression in E. coli. Curr Protoc Protein Sci 2010; 61(1):5.24.1-29.
20. Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 2008; 307(1-2):249-64.
21. Devlin PE, Drummond RJ, Toy P, Mark DF, Watt KW, Devlin JJ. Alteration of amino-terminal codons of human granulocyte-colony-stimulating factor increases expression levels and allows efficient processing by methionine aminopeptidase in Escherichia coli. Gene 1988; 65(1):13-22.
22. Nieuwkoop T, Claassens NJ, van der Oost J. Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design. Microb Biotechnol 2018; 12(1):173-9.
23. Waugh DS. Making the most of affinity tags. Trends Biotechnol 2005; 23(6):316-20.
24. Ramón A, Señorale-Pose M, Marín M. Inclusion bodies: not that bad Front Microbiol 2014; 5:56.
25. Smith DB. Generating fusions to glutathione-S-transferase for protein studies. Methods Enzymol 2000; 326:254-70.
26. Massiah MA, Wright KM, Du H. Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media. Curr Protoc Protein Sci 2016; 84(1):6.13.1-6.
27. Hill CP, Osslund TD, Eisenberg D. The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc Natl Acad Sci U S A 1993; 90(11):5167-71.
28. Bihl MP, Heinimann K, Rudiger JJ, Eickelberg O, Perruchoud AP, Tamm M, et al. Identification of a novel IL-6 isoform binding to the endogenous IL-6 receptor. Am J Respir Cell Mol Biol 2002; 27(1):48-56.
29. Luzina IG, Lockatell V, Lavania S, Pickering EM, Kang PH, Bashkatova YN, et al. Natural production and functional effects of alternatively spliced interleukin-4 protein in asthma. Cytokine 2012; 58(1):20-6.
30. Luzina IG, Keegan AD, Heller NM, Rook GA, Shea-Donohue T, Atamas SP. Regulation of inflammation by interleukin-4: a review of "alternatives". J Leukoc Biol 2012; 92(4):753-64.
31. Luzina IG, Lockatell V, Todd NW, Highsmith K, Keegan AD, Hasday JD, et al. Alternatively spliced variants of interleukin-4 promote inflammation differentially. J Leukoc Biol 2011; 89(5):763-70.
32. Tsytsikov VN, Yurovsky VV, Atamas SP, Alms WJ, White B. Identification and characterization of two alternative splice variants of human interleukin-2. J Biol Chem 1996; 271(38):23055-60.
33. Toghraie FS, Ghaderi A, and Ramezani A. Homology Modeling of an Alternative Splice Variant of Human Granulocyte Colony-Stimulating Factor, G-CSF Isoform D, and Study of Its Binding Properties by Molecular Docking. International Journal of Peptide Research and Therapeutics 2019;1-9.