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Preformulation of New Biological Entities
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Riccardo Torosantucci, Vasco Filipe, Jonathan Kingsbury, Atul Saluja, Yatin Gokarn
In parallel or prior to the biochemical and biophysical characterization to design the formulation space, in silico tools can be used to compute some of the intrinsic physicochemical properties of the candidate under development as well as highlighting spots susceptible to degradation. ExPASy World Wide Web server [168] is a bioinformatics resource portal operated by the Swiss Institute of Bioinformatics, which enables scientists to access a wide range of resources in proteomics, genomics, systems biology, and genetics, besides others. Within the proteomic section, it is possible to predict the expected isoelectric point (pI), molecular weight (MW), secondary structure, and potential protein PTMs based on the primary sequence. Similarly, depending on the primary sequence, several algorithms enable identifying aggregation-prone regions (APRs). Aggrescan, PASTA, SALSA, and TANGO [169] are among the most widely used. Unlike these, spatial aggregation propensities [170] and Aggrescan 3D [171] use a protein 3D structure as an input for the identification of APRs.
Molecular characterization of biosurfactant producing Bacillus cereus strain DRDU1 for its potential application in bioremediation and further EOR studies
Published in Petroleum Science and Technology, 2018
The presence of sfp, sfpO, and arfA genes in the genomic DNA of the isolate XI was evaluated by PCR amplification of the respective gene products, out of which the presence of only sfp gene was confirmed with a length of 650 bp (Supplementary Figure S1). The microbial BLAST analysis of the amplicon showed 99% similarity with Bacillus cereus ATCC 14579 (GenBank accession no. NC_004722.1) as shown in Supplementary Table S3 and Figure S4. The phylogenetic analysis of the sequence with top ten BLAST hits showed 72% similarity with Bacillus cereus strain Rock4–18 (accession number NZ_CM000735.1) at DNA level (Figure 4). The sequence was aligned with available sfp gene sequences of Bacillus family available in NCBI database (Supplementary Table S4) to see its similarity and evolutionary relatedness with these sequences. The phylogenetic tree of the amplified sequence with other sfp gene sequences of Bacillus family shows 100% similarity with Bacillus cereus (YP_083725.1) at nucleotide level (Figure 5) and 79% similarity at amino acid level (Figure 6). GenBank accession number KU721839 was received for the amplified sequence after deposited in NCBI GenBank database. Hypothetical amino acid sequence along with the open reading frames from the amplified sequence (GenBank accession: KU721839) of the isolate Bacillus cereus DRDU1 translated with the help of ExPASy Bioinformatics Resource Portal is shown in Figure 7.
Production of highly soluble native human paraoxonase 2 with potential anti-biofilm property
Published in Preparative Biochemistry & Biotechnology, 2023
Fauzia Parween, Priyamedha Yadav, Kalyani Singh, Rinkoo Devi Gupta
GdnHCl is a mild solubilizing agent.[25] Since active protein was obtained only in GdnHCl buffer, it was used for further solubilizing and purifying the enzyme. 130 ml resuspension buffer per liter culture was used to resuspend the cells. The resuspended cells were kept for 1 hour at room temperature on a rocker. The resuspension buffer was composed of 4.0 ml lysozyme (10 mg/ml), and 1 mM PMSF in 50 mM Tris-HCl pH 8.0. Thereafter, tubes were shaken vigorously after adding 20 ml of 5 M NaCl and incubated on a rocker at room temperature for 30 minutes followed by the addition of 10 ml of 25% Triton X-100. Then the cell lysate was sonicated at 30% amplitude 20 s on/off cycle 5–6 times. The supernatants were collected as washes by centrifuging at 10,000 rpm for 20 minutes at 4 °C. The pellets were resuspended in 100 ml resuspension buffer containing 1 mM PMSF, and 2 ml Triton X-100 (25%) in 50 mM Tris-HCl pH 8.0. This washing step was repeated 4–5 times without Triton X-100. Finally, the pellet was dissolved in IB solubilization buffer containing 4 M GdnHCl, and 10 mM DTT in 100 mM Tris pH 8.1 at a protein concentration of greater than 5 mg/mL approximately, and incubated on the rocker for 2 hours, shaking periodically. The supernatant and pellet (if any) were collected separately after centrifugation at 10,000 rpm for 20 minutes at 4 °C. NanoDrop (ThermoScientific, USA) was used to take the concentration of the proteins. The extinction coefficient for HuPON2 at Abs 0.1% (=1 g/L) is 0.809 assuming all Cys residues be reduced. The physical and chemical parameters of the protein were analyzed using the ProtParam tool of ExPASy (https://web.expasy.org/protparam/) (Table S1).
Characterization of marine bacterial carbonic anhydrase and their CO2 sequestration abilities based on a soil microcosm
Published in Preparative Biochemistry & Biotechnology, 2019
Panchami Jaya, Vinod Kumar Nathan, Parvathi Ammini
CA gene specific primers were used for amplification of CA gene from the isolate AS-75. The PCR reaction was performed using the primer CA-4 F (5′-AGAGAGCATATGAGCGACTTGCAG-3′) and CA-4 R (3′-AGAGAGGGATCCTCAGCAGCAAC-5′) and the resultant PCR amplicon was purified and extracted. Nucleotide Sequence from the sequencing chromatogram were translated into a protein sequence using a Translate tool (http://web.expasy.org). To examine the physiochemical characteristics of the potential isolate, the Expasy ProtParam tool[27] was used and for predicting the secondary structure of protein, the Expasy SOPMA tool[28] were used.