Enrique de-la-Re-Vega

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei.

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.

Recombinant expression of marine shrimp lysozyme in Escherichia coli

Shrimp Lysozyme (Lyz) is a key component of the antibacterial response as part of the innate defense in Crustacea; however, it has not been possible to purify this protein because of the very low amount present in the shrimp blood cells (hemocytes). In an effort to produce enough protein to study its function and biochemical properties we have overexpressed Lysozyme from marine shrimp ( Penaeus vannamei ) in E. coli . A bacterial protein expression system based on the T7 polymerase promoter was used. Although Lyz was produced as insoluble protein in inclusion bodies, its refolding led to an active protein with a yield of ~10%. Details of the protein recombinant expression techniques applied to this shrimp protein are presented.

White Spot Syndrome Virus Orf514 Encodes a Bona Fide DNA Polymerase

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.

Biophysical Characterization Of An Insect Lysozyme From Manduca Sexta

Insect lysozyme from Manduca sexta (MS-lys) was overexpressed in E. coli and refolded to obtain active protein. Recombinant MS-lys presented a globular structure, with an alpha-helical content of 57% as assessed by circular dichroism spectroscopy. Light scattering studies showed that in solution MS-lys has a quasi-monodisperse size distribution, with a rod-like structure similar to nucleation clusters reported in egg lysozyme pre-crystallization stages. These results show that MS-lys is an excellent candidate for crystallization, folding and denaturation studies.

The Pacific oyster (Crassostrea gigas) Hsp70 modulates the Ostreid herpes virus 1 infectivity

Abstract The Ostreid herpes virus type 1 (OsHV‐1) is one of the most devastating pathogen in oyster cultures. Among several factors, as food limitation, oxygen depletion, salinity and temperature variations, episodes of “summer mortality” of the Pacific oyster Crassostrea gigas have also been associated with OsHV‐1 infection. Mortalities of C. gigas spat and juveniles have increased significantly in Europe, and contemporary mortality records of this mollusk in México have been associated with the occurrence of OsHV‐1. In the present study, the expression of the heat shock protein 70 gene from the Pacific oyster correlates with the abundance of DNA polymerase transcripts from the OsHV‐1. This may suggest that the induction on the expression of the Pacific oyster hsp70 may potentially participate in the immune response against the virus. Furthermore, this study reports for the first time a TEM representative image of the OsHV‐1 in aqueous solution, which possesses an icosahedral shape with a diameter of 70 nm × 100 nm. Finally, the examined sequence encoding the ORF4 of the OsHV‐1 isolate from northwest Mexico showed specific sequence variations when compared with OsHV‐1 isolates from distant geographical areas. Graphical abstract Figure. No Caption available. HighlightsWe studied the effect of OsHV‐1 on the expression of C. gigas hsp70.Hsp70 never returned to it amount of mRNA control organism level.Hsp70 levels were related to the infection level.In situ detection of host and pathogen genes in same tissues.First negative aqueous TEM images of the OsHV‐1.

Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a=144.6, b=83.4, c=74.3 Å, β=117.6°. One data set was processed to 3 Å resolution, with an overall Rmeas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen.

De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.