A one-step potentiometric immunoassay for plasma cardiac troponin I using an antibody-functionalized bis-MPA-COOH dendrimer as a competitor with improved sensitivity.

Herein, we have reported a new one-step potentiometric immunoassay for the sensitive and specific detection of human plasma cardiac troponin I (cTnI), a biomarker of cardio-cerebrovascular diseases. Initially, the cTnI biomolecules were immobilized on the surface of a gold nanoparticle-functionalized screen-printed graphite electrode (SPGE). Thereafter, rabbit polyclonal antibodies to cTnI were covalently conjugated to the bis-MPA-COOH dendrimers through typical carbodiimide coupling. The introduction of the target analyte caused a competitive immunoreaction between the immobilized cTnI on the electrode and the conjugated antibody on the dendrimers. The potentiometric measurement was mainly derived from the change in the surface charge on the surface of the modified electrode due to the negatively charged bis-MPA-COOH dendrimers after the immunoreaction. On increasing target cTcI, the number of charged dendrimers on the immunosensor decreased, resulting in a change in the electric potential. Under optimum conditions, the potentiometric immunosensor exhibited good potentiometric responses for the detection of cTcI and allowed the determination of the target analyte at a concentration as low as 7.3 pg mL-1. An intermediate precision of ≤8.7% was accomplished with batch-to-batch identification. Meanwhile, the potentiometric immunosensor showed good anti-interfering capacity and selectivity against other proteins and biomarkers. Importantly, our system displayed high accuracy for the analysis of human plasma serum samples containing target cTcI relative to commercial human cTcI enzyme-linked immunosorbent assay (ELISA) kits.

An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli.

Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc-ß-1,3-Gal-ß-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6-sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.

Application of short hydrophobic elastin-like polypeptides for expression and purification of active proteins.

To investigate the application of short elastin-like polypeptides (ELPs) in the purification of bioactive proteins, short hydrophobic ELP[I] n (n = 30, 40, 50) tags were constructed. Both the ELP[I] n tags and the ELP[I] n -Trx fusion proteins could be stably expressed in Escherichia coli and purified by inverse transition cycling, respectively. Total protein concentrations determined by BCA protein assay showed that the yield of the fusion proteins decreased with increasing ELP length. Measurements of the inverse transition temperature (T t) of the ELP[I] n -Trx under different salts or PEG8000 concentrations showed decreased T t upon elevated concentrations; while, all the T ts were suitable for generating proteins from 4 to 37.5 ºC. Furthermore, to identify a linker peptide for bioactive protein production without the need to remove the ELP[I] n tag, the activity of eGFP protein fused with ELP[I]30 tag by either a poly-N or a G4S linker was quantified using a fluorescence spectrophotometer. The results indicated that the ELP[I]30-eGFP fusion proteins with the poly-N linker showed higher fluorescence levels than those with the G4S linker. Our results demonstrated that short ELP[I] n tags with low T t were useful in protein expression and purification, and poly-N linker played the key role in producing bioactive proteins without the need to remove the ELPs.

Evaluation of the Lyophilized Platelets Function by Thromboelastography in an In Vitro Platelet Transfusion Model.

BACKGROUND: At present, liquid conserved platelets (PLTs) can only be stored at 22°C for up to 5 days. Waste of outdated PLTs and short supply of fresh PLTs were both seen in blood banks. Lyophilized PLTs were considered as one of the candidate replacements for liquid conserved PLTs. It is important to evaluate the function before it can be used in clinical trial. METHODS: In this study, an in vitro platelet transfusion model was established to evaluate the function of rehydrated lyophilized platelets (RLPs) by thromboelastography (TEG). Blood samples from 11 patients were spiked with 3 preparations of specific donors' apheresis platelets (stored at room temperature, frozen, and lyophilized) to an increment equivalent to transfusion with 3x10^11 platelets. Whole blood TEG assay was performed and the maximum amplitude (MA) value was used to evaluate the function of "transfused" platelets. RESULTS: The recovery of rehydrated lyophilized platelets (RLPs) in our study was 81.38% ± 2.38, and mean platelet volume (MPV) was 9.02 ± 0.54 fL. MA was significantly enhanced in the three different groups after the addition of PLTs when compared with the whole blood (WB) group. CONCLUSIONS: Our findings suggest that RLPs are capable of enhancing the MA value as well as fresh and frozen PLTs in vitro. The clinical significance of this remains to be determined.