Isolation of a peptide ligand for affinity purification of factor VIII using phage display

Polypeptides for use in affinity chromatography of factor VIII were identified using phage display technology. Phage libraries were designed to express polypeptide fusions containing five to seven residues flanked by two cysteines that form a disulfide bond. Individual bacteriophage were selected for the ability of these polypeptides to bind factor VIII, and then release the protein under mild elution conditions. Strong consensus sequences were observed that appear to be necessary for this reversible interaction. Chemically synthesized ligands identified by this screening were immobilized onto a chromatographic support and used for affinity purification of factor VIII from a complex feedstream. A chromatographic step was developed that provided a 10000-fold reduction in host cell proteins and DNA, while providing exceptional product recovery.     

Understanding the Hofmeister effect in interactions between chaotropic anions and lipid bilayers: molecular dynamics simulations

A set of all-atom molecular dynamics simulations have been performed to better understand critical phenomena regarding a Hofmeister series of anions and lipid bilayers. The simulations isolate the effect of anion size and show clear differences in the interactions with the dipolar phoshpatidylcholine headgroup. Cl- anions penetrate into the headgroup region of the bilayer, but the simulations confirm theories which predict that larger anions penetrate more deeply, into a more heterogeneous and hydrophobic molecular region. That anion size leads to such differences in partitioning in the bilayer provides atomic-level support to hypotheses inspired by several experimental studies. The ability of larger anions to bury deep within the bilayer is correlated with a less well-structured hydration shell, shedding of which upon penetration incurs a smaller penalty for the larger anions than for Cl-.     

Synthesis of substituted 5-aminomethyl tetrahydro-isoquinolines and dihydro-isoindoles

The synthesis of ten substituted aminomethylene tetrahydro-isoquinolines is described, proceeding in eight steps from 5-hydroxyisoquinoline via reductive amination of N-Boc tetrahydro-isoquinoline 5-carboxaldehyde. Likewise, reductive amination was used to prepare four substituted dihydro-isoindoles from the corresponding aldehyde. The dihydro-isoindole ring system was conveniently accessed via a 2+2+2 cycloaddition reaction.     

Patents and literature

Protein engineering and site-directed mutagenesis is becoming immensely important in both fundamental studies and commercial applications involving proteins and enzymes in biocatalysis. Protein engineering has become a powerful tool to help biochemists and molecular enzymologists elucidate structure-function relationships in enzymic active sites, to understand the intricacies of protein folding and denaturation, and to alter the selectivity of enzymatic catalysis. Commercial applications of engineered enzymes are being developed to increase protein stability, widen or narrow substrate specificity, and to develop novel approaches for use of enzymes in organic synthesis, drug design, and clinical applications. In addition to protein engineering, novel expression systems have been designed to prepare large quantities of genetically engineered proteins. Recent US patents and scientific literature on protein engineering, site-directed mutagenesis, and protein expression systems related to protein engineering are surveyed. Patent abstracts are summarized individually and a list of literature references are given.     

Introduction to Session 5

Applied Biochemistry and Biotechnology -     

Thermal degradation of bisphenol a polycarbonate

Bisphenol A polycarbonate (Makrolon 3000) has been purified and fractionated. Thin films of these samples have been degraded by heating at 200° under continuous evacuation (pressure less than 0- 1 Pa) for periods of several hours. The molecular weight is observed (by gel permeation chromatography) to increase with time at 200°. Eventually gel is formed. The variation of the number- and weight-average molecular weights with heating time and the effect of the initial molecular weights of the polycarbonate samples are consistent, in the main, with the Davis-Golden mechanism of thermal degradation of bisphenol A polycarbonate; i.e. the principal reactions are condensation and trifunctional branching. Chain scission due to hydrolysis of the carbonate linkage is absent under our conditions.     

Crystallinity of ethylene oxide oligomers

Nonaethylene glycol and pentadecaethylene glycol and their dimethyl ethers have been prepared and characterized, with respect to crystallinity by wide- and small-angle X-ray scattering, Raman scattering, i.r. spectroscopy and differential scanning calorimetry. Wide-angle X-ray scattering is similar to that from high molecular weight poly(ethylene oxide). The crystal habit is lamella. The lamellae are highly crystalline and the surface layers are ordered. Comparison with crystalline poly(ethylene oxide) prepared conventionally, and having a distribution of chain lengths, shows that such samples crystallize into lamellae with disordered surface layers.     

Coordinating California’s efforts to promote waste to alcohol production

Alcohol fuels produced from biomass can improve air quality, enhance energy security, create employment opportunities, and reduce waste disposal problems. Opportunities in California exist to produce alcohols from waste streams from various sectors of the economy. Government agencies have promoted waste-to-alcohol activities, but efforts have been inconsistent and intermittent. Often these efforts have been hindered by contradictory but mandate-driven policies. A prudent approach to coordinate statewide efforts includes the development of an integrated statewide policy to examine barriers that impede private sector business efforts to produce alcohols from biomass. A multi-agency task force to promote research, development, commercialization, and marketing efforts for biomass-produced alcohols is desirable. The views and opinions contained in this document do not necessarily reflect those of the California Energy Commission, its staff, management, or the State of California.     

Synthesis and anion binding properties of 2,5-diamidothiophene polypyrrole Schiff base macrocycles

[reaction: see text] Two easy-to-synthesize polypyrrolic 2,5-diamidothiophene Schiff base macrocycles are reported, along with their anion binding properties as determined via UV-vis spectroscopic titrations carried out in dichloroethane. There is a striking difference between the interactions with anions of the two macrocycles, a finding ascribed to differences in their rigidity. For example, the more flexible dipyrromethane-derived macrocycle displays a 1.2:1 hydrogen sulfate versus nitrate selectivity, while its more rigid bipyrrole-derived congener shows a 7.4:1 selectivity in favor to hydrogen sulfate.