Immunological comparison of recombinant shrimp allergen Pen m 4, produced in Pichia pastoris and Escherichia coli.

Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3-2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.

Comparative Analysis of Mesophilic YqfB-Type Amidohydrolases.

The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications.