Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A

The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols.     

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site-specific chemical modification

Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans-splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C-terminal half (C-intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.     

Isopeptide ligation catalyzed by quintessential sortase A: mechanistic cues from cyclic and branched oligomers of indolicidin

The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ε-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (ε-amine) and Thr (α-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.     

葡萄球菌A蛋白基因的克隆及其IgG受体区新型表达载体的构建

旨在克隆SPA基因并将该基因的IgG结合区亚克隆至毕赤酵母表达载体中。以金黄色葡萄球菌CowanI菌株基因组为模板,对葡萄球菌A蛋白(SPA)基因全长序列进行PCR扩增,再将PCR产物克隆入pMD18-T质粒,将DNA测序所得的结果用Blastn软件进行在线同源比对,经鉴定为SPA基因序列后,在线对其进行功能区域(IgG-Fc受体区)的预测,将该区域亚克隆入表达载体pPICZaA。结果显示,从CowanI菌株中成功地扩增到SPA基因,与NCBI中公布的序列同源性高达97%,同时构建了IgG-Fc受体区新型的表达载体     

Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.     

Advantages in using non-isothermal bioreactors in bioremediation of water polluted by phenol by means of immobilized laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilized on a polypropylene membrane chemically modified with chromic acid. Ethylenediamine and glutaraldehyde were used as spacer and bifunctional coupling agent, respectively. Phenol was used as substrate.To know how the immobilization procedures affected the enzyme reaction rate the catalytic behavior of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged: (i) the narrower pH–activity profile of the soluble enzyme in comparison to that of the insoluble counterpart and (ii) the increase in pH and thermal stability of the insoluble enzyme.The laccase catalytic behavior was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increased with the increase of the temperature difference.     

Improvement of antibody immobilization using hyperbranched polymer and protein A

For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 μg mL⁻¹. The detection limit was estimated to be 0.53 μg mL⁻¹. The immunosensor holds good selectivity, sensitivity, and repeatability.     

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.     

Structural comparison in solution of a native and retro peptide derived from the third helix of Staphylococcus aureus protein A,domain B: retro peptides,a useful tool for the discrimination of helix stabilization factors dependent on the peptide chain orientation

A peptide fragment corresponding to the third helix of Staphylococcus Aureus protein A, domain B, was chosen to study the effect of the main‒chain direction upon secondary structure formation and stability, applying the retro‒enantio concept. For this purpose, two peptides consisting of the native (Ln) and reversed (Lr) sequences were synthesized and their conformational preferences analysed by CD and NMR spectroscopy. A combination of CD and NMR data, such as molar ellipcitity, NOE connectivities, Hα and NH chemical shifts, ³J_αN coupling constants and amide temperature coefficients indicated the presence of nascent helices for both Ln and Lr in water, stabilized upon addition of the fluorinated solvents TFE and HFIP. Helix formation and stabilization appeared to be very similar in both normal and retro peptides, despite the unfavourable charge–macrodipole interactions and bad N-capping in the retro peptide. Thus, these helix stabilization factors are not a secondary structure as determined for this specific peptide. In general, the synthesis and confirmational analysis of peptide pairs with opposite main‒chain directions, normal and retro peptides, could be useful in the determination of secondary structure stabilization factors dependent on the direction. © 1997 European Peptide Society and John Wiley & Sons, Ltd.