Identification and Expression of Elastin-like Polypeptides

doi:10.13523/j.cb.2003055

China Biotechnology

2020, Vol. 40

Issue (8): 33-40 DOI: 10.13523/j.cb.2003055

Identification and Expression of Elastin-like Polypeptides

ZHANG Xiao-hang¹,LI Yuan-yuan²,JIA Min-xuan^2,³,GU Qi^2,^4,^**(

)

1 Savaid Medical School, University of Chinese Academy of Sciences, Beijing 101408, China
2 State Key Laboratory of Membrane Biology, Institute of Zoology, CAS, Beijing 100101, China
3 College of Life Science, Northeast Agricultural University, Harbin 150030, China
4 Stem Cell and Regenerative Medicine Institute, CAS, Beijing 100101, China

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Abstract

Organ reconstruction may have severals of requirements for the elasticity, stiffness, and biological activity of the materials due to different applications, but currently many materials are challenging to meet these requirements at the same time. For example, polyethylene glycol diacrylate, a kind of widely used elastic material, do not have high biological activity, while bio-materials such as collagen have poor elasticity. As an elastic functional protein which widely existing in animals, elastin is valued in tissue engineering reconstruction of elastic organs for its special properties that can withstand large deformation without destroying its structure. The amino acid sequence of the elastin-like polypeptide (ELP) designed in this article meets the requirements in the project, excellent biological activity and elasticity, according to the basic repeat unit Val-Pro-Gly-Xaa-Gly and preference and degeneracy of the E coli. condon. After the plasmid was constructed, the ELP was expressed and collected in E coli. BL21 (DE3), and then identified the protein by SDS-PAGE. The modulus data of the protein was tested by rheology, the microstructure of the material system was tested by SEM, the biological activity of the material was tested by cell culture. These methods identified the elastic properties, physical structure and biological activity of materials. They contributed the basis for enhancing the elasticity by crosslinking and its application in tissue engineering and organs reconstruction.

Key words： Elastin-like polypeptides Prokaryotic expression Bio-materials

Received: 23 March 2020 Published: 10 September 2020

ZTFLH:

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Corresponding Authors: Qi GU E-mail: qgu@ioz.ac.cn

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	Xiao-hang ZHANG
	Yuan-yuan LI
	Min-xuan JIA
	Qi GU

Cite this article:

ZHANG Xiao-hang,LI Yuan-yuan,JIA Min-xuan,GU Qi. Identification and Expression of Elastin-like Polypeptides. China Biotechnology, 2020, 40(8): 33-40.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2003055 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I8/33

Fig.1 Schematic diagram and identification of the recombination vector pET-28b-RGD-ELP (a) The plasmid map of pET-28b-RGD-ELP (b) Design strategies for amino acid sequences of RGD-ELP(the L、R、K in different colors have a free amino) (c) Recombinant plasmid digestion, 1: pET-28b-RGD-ELP plasmid; 2: pET-28b-RGD-ELP plasmid digested by MluⅠ and HindⅢ; M: kb ladder marker (d) Alignment between the design amino acid sequence and the sequencing result (red)

Fig.2 Analysis of prokaryotic expression RGD-ELP by SDS-PAGE (a) 1:None-induction supernatant; 2:None-induction pricipitation; 3: Supernatant induced by IPTG; 4: Pricipitation induced by IPTG (b) 1、2、3: Discarded precipitate in the ITC cycles;4、5: Solution of the collected protein

Fig.3 The status of 10% RGD-ELP before and after cross-linking and the rheological test of 10% ELP or gelatin mixed with 5% PEG-NHS (a) 10% RGD-ELP in H₂O (b) 1h after 10% RGD-ELP equal volume mixed with 5% PEG-NHS in H₂O (c) The diagram of cross-linking reaction between RGD-ELP and 4-arm-PEG-NHS (d) Rheological test of 10% RGD-ELP or gelatin equal volume mixed with 5% 4-arm-PEG-NHS

Fig.4 Structure of RGD-ELP and PEG-NHS after crosslinked, SEM (a) The process of sample preparation (b) 5% ELP+10% PEG-NHS,×500, bar: 50μm (c) 10% ELP+10% PEG-NHS,×500, bar: 50μm (d) The magnified SEM image of white rectangle in Figure4(c),×2 000, bar: 10μm (e) The magnified SEM image of white rectangle in Figure4(e),×2 000, bar: 10μm (f) The pore cross section(μm²) of different samples

Fig.5 Results of 3D culture of hMSC cells in the mixtures of RGD-ELP or collagen with PEG-NHS Live/dead staining of hMSCs, under confocal microscope (a) 1% COL and 1% PEG-NHS in the mixture, bar: 100μm (b) 1% RGD-ELP and 1% PEG-NHS in the mixture, bar: 100μm (c) Cell viability of the hMSCs, which was cultured 24h after mixed with the materials


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