设计和人工细胞外基质的构建（AECM）蛋白质<em>大肠杆菌</em>对皮肤组织工程

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

1.克隆重组质粒编码AECM蛋白质设计的功能域（ 例如 ，细胞结合结构域和弹性蛋白样重复序列）的氨基酸序列。设计限制位点侧翼的功能结构域的末端，以促进亚克隆使用自由软件根据软件指令（ 例如 ，http://biologylabs.utah.edu/jorgensen/wayned/ape/）。这里，选择唯一的限制性位点不存在于功能结构域来限制消化到预期位点。选择出现在多克隆位点的宿主载体的（MCS）的限制位点（…

Representative Results

在设计包含弹性蛋白样重复融合蛋白，它维持一个整体弹性内容中，融合蛋白18的足够大的部分是很重要的。这是为了确保该融合蛋白构建体保留其弹性蛋白样特征，以便使用ITC进行纯化。的AECM蛋白质设计和本节中所述的序列被明确取自工作由Tjin 等人14。在这项工作中，三AECM蛋白被成功地克隆到pET22b（+）表达载体。连续结扎首先开始与结扎弹力重复插入到pET载体，随后通…

Discussion

重组蛋白质工程是一种通用的技术来创建使用自下而上的方法新的蛋白质材料。的基于蛋白质的材料可根据感兴趣的应用被设计成具有多种功​​能，量身定做。由于增加的进步中克隆和表达的技术，它已成为比较简单（和成本有效），以在一个可再现的和可扩展的方式创建各种人造蛋白质。弹性蛋白样结构域已在许多人造蛋白质被结合，以用作纯化标记，以及赋予机械性能。包含弹性蛋白样序?…

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