Peptides and proteins are two classes of molecules with attractive possibilities for therapeutic applications. However, the bottleneck for the therapeutic application of many peptides and proteins is their short halflives in vivo, typically just a few minutes to hours. Half-life extension strategies have been extensively studied and many of them have been proven to be effective in the generation of long-acting therapeutics with improved pharmacokinetic and pharmacodynamic properties. In this review, we summarize the recent advances in half-life extension strategies, illustrate their potential applications and give some examples, highlighting the strategies that have been used in approved drugs and for drugs in clinical trials. Meanwhile, several novel strategies that are still in the process of discovery or at a preclinical stage are also introduced. In these strategies, the two most frequently used half-life extension methods are the reduction in the rate of renal clearance or the exploitation of the recycling mechanism of FcRn by binding to the albumin or IgG-Fc. Here, we discuss half-life extension strategies of recombinant therapeutic protein via genetic fusion, rather than chemical conjugation such as PEGylation. With the rapid development of genetic engineering and protein engineering, novel strategies for half-life extension have been emerged consistently. Some of these will be evaluated in clinical trials and may become viable alternatives to current strategies for making next-generation biodrugs.

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Glucagon-like peptide-1 (GLP-1) is considered to be a promising peptide for the treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life of GLP-1 limits its clinical application. Albumin-binding domain (ABD) with high affinity for human serum albumin (HSA) has been used widely for half-life extension of therapeutic peptides and proteins. In the present study, novel GLP-1 receptor agonists were designed by genetic fusion of GLP-1 to three kinds of ABDs with different affinities for HSA: GA3, ABD035 and ABDCon. The bioactivities and half-lives of ABD-fusion GLP-1 proteins with different types and lengths of linkers were investigated in vitro and in vivo. The results demonstrated that ABD-fusion GLP-1 proteins could bind to HSA with high affinity. The blood glucose-lowering effect of GLP-1 was significantly improved and sustained by fusion to ABD. Meanwhile, the fusion proteins significantly inhibited food intake, which was beneficial for T2DM and obesity treatment. The half-life of GLP-1 was substantially extended by virtue of ABD. The in vivo results also showed that a longer linker inserted between GLP-1 and ABD resulted in a higher blood glucose-lowering effect. The fusion proteins generated by fusion of GLP-1 to GA3, ABD035 and ABDCon exhibited similar bioactivities and pharmacokinetics in vivo. These findings demonstrate that ABD-fusion GLP-1 proteins retain the bioactivities of natural GLP-1 and can be further developed for T2DM treatment and weight loss. It also indicates that the ABD-fusion strategy can be generally applicable to any peptide or protein, to improve pharmacodynamic and pharmacokinetic properties.

The herbicide chlorimuron-ethyl has been applied widely for weed control in farmland, especially in soybean fields in China over the past decade, but the chronic effects of this herbicide on soil microorganisms, particularly Pseudomonas spp., is not well understood. Taking a continuously cropped soybean field in the town of Fuyuan-a soybean production base of Heilongjiang Province in Northeast China-as a case study, soil samples were collected from plots having received 0-, 5-, and 10-year applications of chlorimuron-ethyl (30 g active component of chlorimuronethyl/ha/year) to study the abundance and diversity of Pseudomonas spp. Meanwhile, an in vitro assay was used to examine the antifungal activities of isolated Pseudomonas spp. against soil-borne pathogens (Fusarium graminearum, Fusarium oxysporum, and Rhizoctonia solani) causing soybean root rot disease. The production of siderophore, hydrogen cyanide (HCN), and lytic enzymes (cellulase, pectinase, and chitinase) by Pseudomonas spp. was also investigated. With 5-and 10-year chlorimuronethyl application, the numbers of soil Pseudomonas spp. decreased from 121×10 2 CFU/g dry soil in the control to 40×10 2 CFU/g dry soil and 13×10 2 CFU/g dry soil, and the Shannon index values decreased from 6.23 to 3.71 and 1.73, respectively. The numbers of antifungal Pseudomonas spp. also decreased, and the proportions of Pseudomonas spp. with antifungal activities against the different test pathogens altered. All the antifungal Pseudomonas spp. could produce siderophore and HCN but not lytic enzymes. The results suggest that long-term application of chlorimuron-ethyl in continuously cropped soybean field had negative effects on the abundance and diversity of soil Pseudomonas spp., including species with different antifungal activities against pathogens. Siderophore and HCN rather than lytic enzymes formed the antifungal metabolites of Pseudomonas spp., and the number of antifungal Pseudomonas that can produce siderophore and HCN decreased markedly under application of chlorimuron-ethyl, especially after 10-year application.

Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1–2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8–10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins.

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Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.

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