Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories.

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies.

Formulation and evaluation of floating, pulsatile, multiparticulates using pH-dependent swellable polymers.

The objective of this study was to develop a floating, pulsatile, multiparticulate drug delivery system intended for chronopharmacotherapy of arthritis. The floating pulsatile drug delivery has the advantage that a drug can be released in the upper gastrointestinal tract after a definite time period of no drug release, i.e. lag time. Cross-linked beads were prepared using low methoxylated pectin (LM104AS), sodium alginate, and low methoxylated pectin (LM104AS) along with sodium alginate by acid- base reaction during ionotropic gelation. Beads were dried in oven at 50 degrees C for 4 h. Aceclofenac was used as a model drug for encapsulation. Drug loaded multiparticulates were subjected to various characterization and evaluation parameters like entrapment efficiency, surface topography, size analysis and in vitro release study. It was found that calcium pectinate beads show maximum drug entrapment. Hence, pectin containing formulation was further studied for buoyancy, DSC and radio imaging study. Drug release study was performed in acidic environment using pH 1.2 buffer solution for 6 h and then at 7.4 pH for 60 min. The total drug release ranges from 5-10% and 90-94% in acidic and basic media, respectively.