Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies

Sustainable Agriculture Reviews 43: Pharmaceutical Technology for Natural Products Delivery Vol. 1 Fundamentals and Applications

International audienceThis edited book comprises of eight chapters dealing on various aspects of pharmaceutical technology for delivery of natural products. Book chapters deal with the solubility and bioavailability enhancement technologies for natural products. Emphasis has also been given on the significance of delivery strategies for improving the therapeutic efficacy of paclitaxel, galantamine and tea constituents

Sustainable Agriculture Reviews 44: Pharmaceutical Technology for Natural Products Delivery Vol. 2 Impact of Nanotechnology

International audienceThis book covers nanotechnology based approaches for improving the therapeutic efficacy of natural products. It critically explores lipid nanoarchitectonics, inorganic particles and nanoemulsion based tools for delivering them. With its chapters from eminent experts working in this discipline, it is ideal for researchers and professionals working in the area

Dog zona pellucida glycoprotein-3 (ZP3): expression in Escherichia coli and immunological characterization

An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplifiedSacI andPstI restricted fragment was cloned in-frame downstream of the T5 promoter underlacoperator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein inEscherichia coli. Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-β-D-thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3β but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs

Optimization and Designing of Amikacin-loaded Poly d, l-Lactide-co-glycolide Nanoparticles for Effective and Sustained Drug Delivery

Purpose: Amikacin, a water-soluble aminoglycoside antibiotic used to treat gram-negative bacillary infections, is a Biopharmaceutics Classification System class III drug having poor permeability and short half-life. It is given parenterally, which limits its use in patients warranting “at-home care.” An oral drug delivery of amikacin is, therefore, imminent. Aim: This work focused on establishing poly d, l-lactide-co-glycolide (PLGA)-based nanoparticles of amikacin with consolidated pharmaceutical attributes capable of circumventing gastrointestinal tract membrane barriers and promoting oral administration of the drug. The partied attributes are suggestive of enhanced uptake of the drug via Peyer’s patches overlaying small intestine and support successful oral delivery. Materials and Methods: To have a robust delivery system, a statistical Box–Behnken experimental design was used and formulation parameters such as homogenization time, probe sonication time, and drug/polymer ratio of amikacin-loaded PLGA nanoparticles (A-NPs) for obtaining monodispersed nanoparticles of adequate size and high drug loading were optimized. Results: The model suggested to use the optimum homogenization time, probe sonication time, and drug/polymer ratio as 30s, 120s, and 1:10, respectively. Under these formulation conditions, the particle size was found to be 260.3nm and the drug loading was 3.645%. Conclusion: Biodegradable PLGA nanoparticulate systems with high payload, optimum size, and low polydispersity index will ensure successful uptake and ultimately leading to better bioavailability. Hence, under the aforementioned optimized conditions, the A-NPs prepared had particle size of 260.3nm, which is appreciable for its permeability across small intestine, and drug loading of 3.645%

Purification, refolding, and characterization of recombinant LHRH-T multimer

To make the native LHRH immunogenic, a multimer of LHRH interspersed with T non-B peptides (r-LHRH-d2) was expressed as recombinant protein in Escherichia coli. The expression level of the recombinant protein was around 15% of the total cellular protein and it aggregated as inclusion bodies. Inclusion bodies from the bacterial cells were isolated and purified to homogeneity. Instead of high concentrations of chaotropic agents, r-LHRH-d2 was solubilized in 50 mM citrate buffer at pH 3 containing 2 M urea. The protein was refolded by 5-fold dilution (pulsatile) with cold 10 mM citrate buffer at pH 6 in presence of 0.3 M L-arginine. Purification of r-LHRH-d2 was carried out by successive passages on CM-Sepharose column at pH 6.0 which retained extraneous proteins and pH 4.8 at which r-LHRH-d2 bound to the resin. The elution was carried out by using linear salt gradient (0.1-1 M NaCl). The overall yield of the purified r-LHRH-d2 was 40% of the initial inclusion body proteins. The purity and homogeneity were confirmed by a single homogeneous peak on analytical HPLC eluting out at 29.51 min and by single band on SDS-PAGE reactive with polyvalent anti-LHRH antibodies. Mass spectroscopic analysis indicated the protein to be of 16.6 kDa which equals the theoretically expected mass. The N-terminal amino acid analysis of r-LHRH-d2 showed the sequence which corresponded to the designed protein. The CD spectrum of the refolded r-LHRH-d2 showed that the multimer has considerable β sheet structure like the monomeric LHRH protein

Amino acid sequence of hGH (Swiss-Prot, P01241).

<p>Bold and italics regions indicate presence of hydrophobic residues involves in making hydrophobic and amphipathic helices.</p

Proteinase K degradation profiles of hGH and asparaginase inclusion bodies isolated after 4 hours of induction.

<p>(<b>a</b>) Rate of degradation of IBs with time. (<b>b</b>) Rate of change in degradation rate of IBs with decrease in turbidities.</p

Solubilization profiles of inclusion bodies isolated from cell harvested at different time points after induction.

<p>(<b>a</b>) hGH IBs (<b>b</b>) Asparaginase IBs. Colored bars indicate the harvesting time.</p