Fusion protein linkers: Property, design and functionality

Abstract

As an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. This review covers the current knowledge of fusion protein linkers and summarizes examples for their design and application. The general properties of linkers derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing the free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles.

Introduction

As a product of recombinant DNA technology, fusion proteins have been developed as a class of novel biomolecules with multi-functional properties. By genetically fusing two or more protein domains together, the fusion protein product may obtain many distinct functions derived from each of their component moieties. Besides their wide applications in biological research such as protein purification [1] and imaging [2], recombinant fusion proteins have also become an important category of biopharmaceuticals (Fig. 1) [3], [4]. For example, many protein drugs are fused to Fc domains of antibodies, such as Fc-immunoglobulin G1 (Fc-IgG1), or to carrier proteins such as human serum albumin (HSA) or transferrin (Tf) to extend their plasma half-lives and to achieve enhanced therapeutic effects [5], [6], [7], [8]. They have also been widely applied for drug targeting, since proteins such as single chain antibodies or ligands for cell surface receptors can specifically target a linked functional protein (e.g. toxin or cytokine) to a specific type of cells [9], [10]. In drug delivery, the conjugation of protein drugs to carrier moieties such as cell penetrating peptides, antibodies or Tf can achieve efficient transport of the protein drugs across biological barriers such as cell membranes, the blood brain barrier or intestinal epithelium [11], [12], [13]. Several fusion proteins drugs including Enbrel® (tumor necrosis factor/Fc-IgG1), Ontak® (interleukin-2/diphtheria toxin), Orencia® (cytotoxic T-lymphocyte antigen-4/Fc-IgG1), Amevive® (leukocyte function antigen-3/Fc-IgG1), Arcalyst® (interleukin-1 receptor extracellular domain/ Fc-IgG1), and Nplate® (thrombopoietin/Fc-IgG1) have been approved by the FDA [14], [15], [16]. With the rapid advancement of biotechnology, it is foreseeable that fusion protein technology will have increasing importance in creating novel protein therapeutics and in improving the performance of current protein drugs.

The successful construction of a recombinant fusion protein requires two indispensable elements: the component proteins and the linkers. The choice of the component proteins is based on the desired functions of the fusion protein product and, in most cases, is relatively straightforward. On the other hand, the selection of a suitable linker to join the protein domains together can be complicated and is often neglected in the design of fusion proteins. Direct fusion of functional domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins [17], low yield in protein production [18], or impaired bioactivity [19], [20]. Therefore, the selection or rational design of a linker to join fusion protein domains is an important, yet underexplored, area in recombinant fusion protein technology.

This review will summarize the current knowledge of linker design in recombinant fusion proteins. First, an overview of the properties of linkers in naturally-occurring multi-domain proteins will be provided as a general reference for linker design. Next, the empirical linkers that have been applied to the successful construction of recombinant fusion proteins will be discussed along with examples. Lastly, various functions that can be achieved by utilizing linkers in recombinant fusion proteins will be presented, including improving folding and stability, facilitating protein expression, increasing the intrinsic biological activities, enabling targeting toward specific sites in vivo, and altering the pharmacokinetic (PK) profiles of fusion proteins.