Volume 25, Issue 5 (Jul-Aug 2017)
JSSU 2017, 25(5): 361-370 |
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Rezaeinasab N, Zeinoddini M, Saeeidinia A. Cloning, expression and functional assessment of elastin like protein: a new protein in medical engineering
. JSSU 2017; 25 (5) :361-370
URL: http://jssu.ssu.ac.ir/article-1-4033-en.html
URL: http://jssu.ssu.ac.ir/article-1-4033-en.html
Abstract: (4851 Views)
Introduction: Elastin like protein or ELP is a synthetic biopolymer consisting the pentapeptide repeats of VPGXG (X can be any amino acid except Pro). This protein is the thermal responsive polypeptide that undergoes a reversible phase transition. At a temperature below the transition temperature (Tt), ELP molecules assume an extended conformation and thus are soluble in aqueous solution; but upon the temperature shift higher than the Tt, however, ELPs become insoluble and form the segregated phase prone to ‘aggrigate’ form. The aim of this study was cloning, expression and activity of ELP60 according to Tt.
Methods: Firstly, ELP10 (150 bp) was synthesized and cloned into pBluescript. Then, Elp60 (900 bp) was produced using recursive directional ligation (RDL). Finally, ELP60 was subcloned into modified pET25 and ELP60 expression was confirmed using SDS-PAGE method. Also, ELP60 were purified according to Tt and performance of inverse transition cycle (ITC), which confirmed its activity.
Results: The results were shown that the pET-ELP60 constructed and ELP60 expressed; it was successfully purified (about 90%) in one step and non-chromatographic method.
Conclusion: From produced recombinant protein can be used for simple and easy purification of the recombinant protein as pharmaceutical drug, smart drug delivery and tissue engineering
Methods: Firstly, ELP10 (150 bp) was synthesized and cloned into pBluescript. Then, Elp60 (900 bp) was produced using recursive directional ligation (RDL). Finally, ELP60 was subcloned into modified pET25 and ELP60 expression was confirmed using SDS-PAGE method. Also, ELP60 were purified according to Tt and performance of inverse transition cycle (ITC), which confirmed its activity.
Results: The results were shown that the pET-ELP60 constructed and ELP60 expressed; it was successfully purified (about 90%) in one step and non-chromatographic method.
Conclusion: From produced recombinant protein can be used for simple and easy purification of the recombinant protein as pharmaceutical drug, smart drug delivery and tissue engineering
Keywords: Cloning, Elastin Liked Protein, Inverse Transition Cycling, Purification, Recursive Directional Ligation.
Type of Study: Original article |
Subject:
Biochemistry
Received: 2016/12/29 | Accepted: 2017/07/9 | Published: 2017/10/2
Received: 2016/12/29 | Accepted: 2017/07/9 | Published: 2017/10/2
References
1. Kowalczyk T, Hnatuszko-Konka K, Gerszberg A, Kononowics AK. Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers. World J Microbiol Biotechnol 2014; 30(8): 2141-52.
2. Roberts S, Dzuricky M, Chilkoti A. Elastin-like polypeptides as models of intrinsically disordered proteins. FEBS Lett 2015; 589(19): 2477-86.
3. Li B, Alonso DO, Daggett V. The molecular basis for the inverse temperature transition of elastin. J Mol Biol 2001; 305(3): 581-92.
4. Park JE, Won JI. Thermal behaviors of Elastin- like Polypeptides (ELPs) according to their physical properties and environmental conditions. Biotechnol Biopro Eng 2009; 14: 662-67.
5. Ribeiro A, Arias FJ, Reguera J, Alonso M, Rodriguze-Cabello JC. Influence of the amino-acid sequence on the inverse temperature transition of elastin-like polymers. Biophys J 2009; 97(1): 312-20.
6. Gao W, Xu D, Lim DW, Craig SL, Chilkoti A. In situ growth of a thermoresponsive polymer from a genetically engineered elastin-like polypeptide. Poltm Chem 2011; 2: 1561-66.
7. Christensen T, Amiram M, Dagher S, Trabbic-Carlson K, Shamji MF, Setton LA, et al. Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins. Protein Sci 2009; 18(7): 1377-87.
8. Meyer DE, Chilkoti A. Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides. Biomacromulecules 2004; 5(3): 846-51.
9. Meyer DE, Chilkoti A. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation : examples from the elastin-like polypeptide system. Biomacromol 2002; 3(2): 357-67.
10. McDaniel JR, MacKay JA, Quiroze FG, Chilkoti A. Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes. Biomacromol 2010; 11(4): 944-52.
11. Sallach RE, Conticello VP, Chaikof EL. Expression of a recombinant elastin-like protein in pichia pastoris. Biotechnol Prog. 2009; 25(6):1810-18.
12. Conley AJ, Joensuu JJ, Jevnikar AM, Menassa R, Brandle JE. Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants. Biotechnol Bioeng 2009; 103(3):562-73.
13. Trabbic-Carlson K, Liu L, Kim B, Chilkoti A. Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Sci 2004; 13(12):3274-84.
14. Arias FJ, Santos M, Fernandez-Colino A, Pinedo G, Girotti A. Recent contributions of elastin-like recombinamers to biomedicine and nanotechnology. Curr Top Med Chem 2014; 14(6):819-36.
15. Simnick AJ. Biomedical and biotechnological applications of elastin like polypeptides. Polymer Rev 2007; 47(1): 121-54.
16. Ghandehari H, Cappello J. Genetic engineering of protein-based polymers: Potential in controlled drug delivery - Commentary. Pharm Res 1998; 15:813–815.
17. Coolbaugh MJ, Shakalli Tang MJ, Wood DW. High-throughput purification of recombinant proteins using self-cleaving intein tags. Anal Biochem 2017; 516: 65-74.
18. Chow DC, Dreher MR, Trabbic-Carlson K, Chilkoti A. Ultra-high expression of a thermally responsive recombinant fusion protein in E-coli. Biotechnol Prog 2006; 22:638–646.
19. Urry DW, Parker TM, Reid MC, Gowda DC. Biocompatibility of the bioelastic materials, poly(Gvgvp) and its gamma-irradiation cross-linked matrix - summary of generic biological test results 1991; 6(3): 263-82.
20. Urry DW, Peng S, Parker T. Delineation of electrostatic- and hydrophobic-induced pka shifts in polypentapeptides: the glutamic acid residue. J Am Chem Soc 1993; 115:7509–7510.
21. Nagapudi K, Brinkman WT, Leisen JE, Huang L, McMillan RA, Apkarian RP, Conticello VP, Chaikof EL. Photomediated solid-state cross-linking of an elastin-mimetic recombinant protein polymer. Macromolecules 2002; 35:1730–1737.
22. Urry DW, Hayes LC, Gowda DC, Harris CM, Harris RD. Reduction-driven polypeptide folding by the delta-TT mechanism. Biochem Biophys Res Commun. 1992; 188:611–617
23. Walkera L, Perkins E, Kratzb F, Raucher D. Cell penetrating peptides fused to a thermally targeted biopolymer drug carrier improve the delivery and antitumor efficacy of an acid-sensitive doxorubicin derivative. Int J Pharm 2012; 436: 825-32.
24. MacEwan SR, Chilkoti A. Applications of elastin like polypeptides in drug delivery. J Con Rel 2014; 190: 314-30.
25. Nettles DL, Chilkot A, Setton LA. Applications of Elastin-like Polypeptides in Tissue Engineering. Adv Drug Deliv Rev 2010; 62: 1479-85.
26. Hassouneh W, Christensen T, Chilkoti A. Elastin-like Polypeptides as a Purification Tag for Recombinant Proteins. Curr Protoc Protein Sci 2010; Chapter 6: Unit 6.11.
27. Banki MR, Feng LA, Wood DW. Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat Methods 2005; 2: 659-61.
28. Meyer DE, Chilkoti A. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 1999; 17(11): 1112–15.
29. Floss DM, Schallau K, Rose-John S, Conrad U, Scheller J. Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical applicatiom. Trends Biotechnol 2010; 28(1): 37-45.
30. Fong BA, Wood DW. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation. Microb Cell Fact 2010; 9:77.
31. Fong BA, Gillies AR, Ghazi I, LeRoy G, Lee KC, Westblade LF, et al. Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag. Protein Sci 2010; 19(6): 1243-52.
32. McPherson DT, Xu J, Urry DW. Product purification by reversible phase transition following Escherchia coli expression of genes encoding up to 251 repeats of the elastromeric pentapeptide GVGVP. Protein Expr Purif 1996; 7(1): 51-7.
33. Fong BA, WY W, Wood DW. Optimizing of ELP –intein mediated protein purification by salt substitution. Protein Expr Purif 2009; 66(2): 198-202.
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