In-vivo half-life and hypoglycemic bioactivity of a fusion protein of exenatide and elastin-based polypeptide from recombinant Saccharomyces cerevisiae.

Exenatide (Ex) is a 39-amino acid peptide of glucagon-like peptide-1 (GLP-1) receptor agonist that was approved by the FDA in 2005 as a Type II diabetes treatment. It shows a 53% homology with GLP-1 but has an extended half-life (ca. 2.4 h) relative to GLP-1 (ca. 2-3 min). In this study, to further extend its in vivo half-life, we constructed a fusion protein (Ex-(EBP)10-6xHis) using a biocompatible and inert elastin-based polypeptide (EBP) as a fusion partner. Valine was inserted into the guest position of the pentapeptide (VPGXG), no linker sequence was inserted in between the EBPs, and (EBP)10-6xHis tag was attached to the C-terminus of exenatide. By using a recombinant Saccharomyces cerevisiae expression system, the fusion protein was expressed and secreted to the broth and purified by Ni-NTA affinity chromatography. Compared with the native exenatide, the physical half-life of the fusion protein was ca. 3.7-fold extended while approximately 72% of the in-vitro insulin secreting activity was maintained. However, the biological half-life measured by a glucose tolerance test (GTT) and the hypoglycemic test in mice was not significantly different from that of the native form. The effects of EBPylation on bioactivity and half-life of the fusion protein are similar to those of PEGylation. The result suggests that the bioactivity and half-life should be carefully balanced to obtain optimal fusion proteins. We expect that EBPylation using an optimal repeat number of EBP can be an alternative to chemical modification for therapeutic biobetters with extended half-life.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Carbon nanotube-assisted enhancement of surface plasmon resonance signal.

We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM-CSF (0.1-1,000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production.