Expression in Trichoplusia ni (High Five) insect cells and partial purification of a chimeric version of active human factor VIII

Introduction/Objectives: Purification of recombinant Factor VIII from mammalian cells poses remarkable challenge, because of its suboptimal yield and significant1y high cost. Bacalovirus derived expression in insect cells is an appropiate system to overcome these limitations. However, several reports have concluded that Sf9 cells are not suitable for expression of structurally correct factor VIII (fVIII). We have developed a cDNA construct encoding chimeric, full-length human fVIII in which dispensable B-domain has been replaced with significantly shorter B-domain derived from its ortholog in Fugu rubripe. This cDNA was successfully applied for expression and purificacion of active fVIII protein in Trichoplusia ni insect cells, known as High Five (Invitrogen)

Expression in Trichoplusia ni (High Five) insect cells and partial purification of a chimeric version of active human factor VIII

Introduction/Objectives: Purification of recombinant Factor VIII from mammalian cells poses remarkable challenge, because of its suboptimal yield and significant1y high cost. Bacalovirus derived expression in insect cells is an appropiate system to overcome these limitations. However, several reports have concluded that Sf9 cells are not suitable for expression of structurally correct factor VIII (fVIII). We have developed a cDNA construct encoding chimeric, full-length human fVIII in which dispensable B-domain has been replaced with significantly shorter B-domain derived from its ortholog in Fugu rubripe. This cDNA was successfully applied for expression and purificacion of active fVIII protein in Trichoplusia ni insect cells, known as High Five (Invitrogen)

Spectroscopic Investigation of Stellacyanin Mutants: Axial Ligand Interactions at the Blue Copper Site

Detailed electronic and geometric structural descriptions of the blue copper sites in wild-type (WT) stellacyanin and its Q99M and Q99L axial mutants have been obtained using a combination of XAS, resonance Raman, MCD, EPR, and DFT calculations. The results show that the origin of the short Cu−S(Cys) bond in blue copper proteins is the weakened axial interaction, which leads to a shorter (based on EXAFS results) and more covalent (based on S K-edge XAS) Cu−S bond. XAS pre-edge energies show that the effective nuclear charge on the copper increases going from O(Gln) to S(Met) to no axial (Leu) ligand, indicating that the weakened axial ligand is not fully compensated for by the increased donation from the thiolate. This is further supported by EPR results. MCD data show that the decreased axial interaction leads to an increase in the equatorial ligand field, indicating that the site acquires a more trigonally distorted tetrahedral structure. These geometric and electronic structural changes, which result from weakening the bonding interaction of the axial ligand, allow the site to maintain efficient electron transfer (high HDA and low reorganization energy), while modulating the redox potential of the site to the biologically relevant range. These spectroscopic studies are complemented by DFT calculations to obtain insight into the factors that allow stellacyanin to maintain a trigonally distorted tetrahedral structure with a relatively strong axial Cu(II)−oxygen bond

Electron-transfer rates govern product distribution in electrochemically-driven P450-catalyzed dioxygen reduction

Developing electrode-driven biocatalytic systems utilizing the P450 cytochromes for selective oxidations depends not only on achieving electron transfer (ET) but also doing so at rates that favor native-like turnover. Herein we report studies that correlate rates of heme reduction with ET pathways and resulting product distributions. We utilized single-surface cysteine mutants of the heme domain of P450 from Bacillus megaterium and modified the thiols with N-(1-pyrene)-iodoacetamide, affording proteins that could bond to basal-plane graphite. Of the proteins examined, Cys mutants at position 62, 383, and 387 were able to form electroactive monolayers with similar E_(1/2) values (− 335 to − 340 mV vs AgCl/Ag). Respective ET rates (k_so) and heme-cysteine distances for 62, 383, and 387 are 50 s^(-1) and 16 Ǻ, 0.8 s^(–1) and 25 Ǻ, and 650 s^(–1) and 19 Ǻ. Experiments utilizing rotated-disk electrodes were conducted to determine the products of P450-catalyzed dioxygen reduction. We found good agreement between ET rates and product distributions for the various mutants, with larger k_so values correlating with more electrons transferred per dioxygen during catalysis