Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC) to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer.

BACKGROUND: The low stability of gammaretroviral and lentiviral vectors affects their production, making high quality clinical preparations a difficult goal to achieve. Recently, our laboratory has shown that the main inactivation mechanism for both these vectors is the loss of their capacity to perform reverse transcription. The present study aimed to increase the stability of gammaretroviral and lentiviral at 37 degrees C and at 4 degrees C. METHODS: Inactivation studies were performed with gammaretroviral and lentiviral vectors at 37 and 4 degrees C, with and without several stabilizing compounds. The residual viral infectivity and reverse transcription capacity of these samples were tested. RESULTS: The results obtained demonstrate that it is possible to increase the stability of reverse transcription and the infectivity stability of purified gammaretroviral vectors by adding recombinant human albumin (rHSA) to the storage buffer, both at 37 degrees C and at 4 degrees C. For lentiviral vectors, it was observed that further protection was needed. This was achieved by adding lipids to the storage buffer, using a mixture of lipoproteins and rHSA. The difference of stabilization between gammaretroviral and lentiviral vectors was validated by performing stabilization tests with vectors possessing different envelope proteins and produced by different cell lines. CONCLUSIONS: The presented study reveals that it is possible to increase the half-life of purified gammaretroviral and lentiviral vectors at 37 degrees C and at 4 degrees C, but the two vectors have different stabilization requirements: for retroviral vectors, the addition of rHSA is enough and, for lentiviral vectors, it is necessary to add both lipoproteins and rHSA. The increase of the stability of the reverse transcription process was shown to have a high impact with respect to the increase of the stability of infectivity.

Characterization of a highly thermostable extracellular lipase from Lactobacillus plantarum.

After screening for the presence of lipase activity in lactobacilli isolated from "chouriço", a traditional Portuguese dry fermented sausage, a strain of Lactobacillus plantarum (DSMZ 12028) was chosen for extracellular lipase characterisation and purification. Proteinase K did not significantly affect lipolytic activity, as opposed to trypsin, which completely eliminated this activity. Among NaCl, Ca2+, EDTA, BSA, glycerol, Mn2+ and Mg2+, only Mn2+ and Mg2+ stimulated the lipase. Purification by gel filtration chromatography and gel electrophoresis revealed four bands, between 98 and 45 kDa, all with lipolytic activity against olive oil.