설계 및 인공 세포 외 기질의 건설 (aECM) 단백질에서<em> 대장균</em> 피부 조직 공학

Summary

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Abstract

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Introduction

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering^1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins^3,4.

Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications^5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair^2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic⁵.

Artificial extracellular matrix proteins have been developed for tissue engineering applications^8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials¹². Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins¹⁰. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)¹³. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities⁸.

Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers¹⁴. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin^2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin α₃Β₁ has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)^16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.

The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)₂VPGKG(VPGIG)₂}₈ that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films¹². Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)¹⁴. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (T_t)^18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique²¹.

In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

Protocol

aECM 단백질 재조합 플라스미드 인코딩 1. 복제 기능 도메인 (예, 세포 결합 도메인과 엘라스틴 같은 반복)의 아미노산 서열을 디자인. 기능 영역의 단부의 측면에 디자인 제한 부위는 서브 클로닝 소프트웨어 명령에 따른 무료 소프트웨어를 사용하여 (예 http://biologylabs.utah.edu/jorgensen/wayned/ape/)을 용이하게한다. 여기서, 의도 사이트로 소화를 한정하기 위해 기능 도메인에 존재?…

Representative Results

엘라스틴 형상의 반복을 포함하는 융합 단백질을 설계에서, 전체 콘텐츠 엘라스틴, 융합 단백질 (18)의 충분한 부분을 유지하는 것이 중요하다. 이 융합 단백질 작 제물이 정제를 위해 ITC를 사용하기 위해, 그것의 엘라스틴과 같은 특성을 보유하고 있는지 확인하는 것이다. aECM 단백질 설계 및이 절에서 설명하는 순서는 특히 Tjin 등. (14)에 의해 직장에서 촬영되었다. ?…

Discussion

재조합 단백질 공학은 상향식 접근을 사용하여 신규 한 단백질 물질을 생성하는 다용도 기술이다. 단백질 계 물질은 관심있는 용도에 따라, 복수의 기능을 갖도록 설계 맞추어 질 수있다. 인해 복제 및 단백질 발현을 증가시키는 기술의 발전에, 그것을 재현하고 확장 가능한 방식으로 인공 단백질의 다양성을 생성하기 위해 상대적으로 간단한 (그리고 비용 효율)이되었다. 엘라스틴 유사 도메인?…

Materials

pET22b (+)	Novagen	69744	T7 expression vectors with resistance to ampicillin
BL21(DE3)pLysS	Invitrogen	C6060-03	additional antibiotics – chloramphenicol
Isopropyl-beta-D-thiogalactoside (IPTG)	Gold Biotechnology	I2481C	1M stock solution with autoclaved water, make fresh prior to induction.
QIAprep Spin Miniprep Kit	Qiagen	27106	plasmid isolation kit
T4 ligase	New England Biolabs	M0202S
Ampicillin	Affymetrix	11259
Chloramphenicol	Affymetrix	23660
Zymoclean™ gel DNA recovery kit	Zymo Research	D4001
XL10-gold strain	Agilent Technologies	200315