Enhancement of recombinant soluble dengue virus 2 envelope domain III protein production in Escherichia coli trxB and gor double mutant

The dengue virus is currently the most important flavivirus causing human diseases in the tropical and subtropical regions of the world. The envelope protein domain III of dengue virus type 2 (D2EIII), which induces protective and neutralizing antibodies, was expressed as an N-terminal fusion to a hexa-histidine tag in Escherichia coli. The expression of recombinant D2EIII of 103 amino acids in the soluble form can be achieved using suitable host strains, such as Origami, at a low induction temperature of 18 degrees C. The enhanced production of the soluble protein could be attributed to the thioredoxin reductase (trxB) and glutathione reductase (gor) double mutations in the Origami genome. The soluble and refolded D2EIII proteins were recognized by different antibodies including human patient antiserum. The immunization of rats with soluble D2EIII protein elicited the production of antibodies that could recognize the D2EIII protein in the D2EIII precursor protein and in C-terminal truncated dengue envelope protein type 1-4. Thus, this protein production system is suitable for the production of authentic recombinant dengue proteins that may be used in the diagnosis of the dengue virus infection or in vaccine development.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.