BackgroundSilk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.ResultsThis study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.ConclusionsWe have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.

The existing data support Portugal as the western European country with the highest HIV-1 subtype diversity. However, detailed phylogenetic studies of Portuguese HIV-1 epidemics are still scarce. Thus, our main goal was to analyze the phylodynamics of a local HIV-1 infection in the Portuguese region of Minho. Molecular epidemiological analysis was applied to data from 289 HIV-1-infected individuals followed at the reference hospital of the province of Minho, Portugal, at which isolated viruses had been sequenced between 2000 and 2012. Viruses of the G (29.1%) and B (27.0%) subtypes were the most frequent, followed by recombinant forms (17.6%) and the C (14.5%), F1 (7.3%), and A1 (4.2%) subtypes. Multinomial logistic regression revealed that the odds of being infected with the A1 and F1 subtypes increased over the years compared with those with B, G, or C subtypes or recombinant viruses. As expected, polyphyletic patterns suggesting multiple and old introductions of the B and G subtypes were found. However, transmission clusters of non-B and non-G viruses among native individuals were also found, with the dates of the most recent common ancestor estimated to be in the early 2000s. Our study supports that the HIV-1 subtype diversity in the Portuguese region of Minho is high and has been increasing in a manner that is apparently driven by factors other than immigration and international travel. Infections with A1 and F1 viruses in the region of Minho are becoming established and are mainly found in sexually transmitted clusters, reinforcing the need for more efficacious control measures targeting this infection route.

a b s t r a c tBackground: In 2006, bronchiolitis due to adenovirus nosocomial infections resulted in the closure of a pediatric department in northern Portugal. Objectives: To determine the etiology of bronchiolitis in northern Portugal. Study design: It was a prospective multicenter study on the etiology of bronchiolitis during the respiratory syncytial virus (RSV) season (November-April). Children ≤24 months of age admitted for a first wheezing episode were included. Nasopharyngeal specimens were analyzed by an indirect immunofluorescentantibody assay (IFA) for RSV, adenovirus (HAdV), parainfluenza (PIV) 1-3 and influenza (IV) A and B and by polymerase chain reaction (PCR) or reverse transcription-PCR for the same viruses and for human metapneumovirus (hMPV), bocavirus (HBoV), rhinovirus (HRV), coronaviruses (229/E; NL63; OC43; HKU1) and enterovirus. Results: During this period, 253 children were included, 249 IFA analyses and 207 PCRs were performed. IFA detected RSV in 58.1%; PCR increased it to 66.7%. IFA detected HAdV in 3.2%, PCR 10.0%. PCR detected IV A in 5; IV B in 2; PIV 1 in 6, PIV 2 in 4 and PIV 3 in 11 cases. HBoV, as single agent in 2 cases, and HRV were positive in 8 samples and hMPV in 11. With this virus panel, 19.7% remained without etiology. Conclusions: The most frequent agent was RSV, followed by HAdV. PCR can be cost-effective and more accurate than IFA, which is crucial for HAdV that may be associated with significant mortality (IFA alone did not detect 2/3 of the cases).

Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications, yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L(-1) with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high preinduction growth rates and low inducer and oxygen concentrations are allowed whereas reduced postinduction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible, and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace.

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