A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII

We have constructed new B domain deletion derivatives of human factor VIII (FVIII) by manipulating the cDNA using recombinant DNA techniques. One of these new derivatives, FVIII delta II, in which amino acids 771(pro)-1666(asp) have been deleted, no longer contains the protease cleavage site at amino acid position 1648(arg)-1649(glu) known to be involved in the initial step of FVIII processing. We have expressed this molecule in both baby hamster kidney (BHK) 21 cells using the vaccinia virus (VV) expression system and have established Chinese hamster ovary (CHO) derived permanent cell lines expressing either recombinant (r)FVIII or FVIII delta II. The characteristics of FVIII delta II have been compared to those of rFVIII and/or plasma derived (pd) FVIII. FVIII delta II has the following properties: (i) it exhibits FVIII procoagulant activity; (ii) it is expressed at 5-fold higher levels than is the complete molecule in comparable systems; (iii) it migrates for the most part as a single major band on SDS-PAGE, in contrast to the complete molecule; (iv) it is activated to a greater extent by thrombin than is either rFVIII or pdFVIII; and (v) it retains the ability to bind von Willebrand factor (vWf).     

Social effects on fruit fly courtship song

Courtship behavior in Drosophila has often been described as a classic innate behavioral repertoire, but more recently extensive plasticity has been described. In particular, prior exposure to acoustic signals of con‐ or heterspecific males can change courtship traits in both sexes that are liable to be important in reproductive isolation. However, it is unknown whether male courtship song itself is socially plastic. We examined courtship song plasticity of two species in the Drosophila melanogaster subgroup. Sexual isolation between the species is influenced by two male song traits, the interpulse interval (IPI) and sinesong frequency (SSF). Neither of these showed plasticity when males had prior experience of con‐ and heterospecific social partners. However, males of both species produced longer bursts of song during courtship when they were exposed to social partners (either con‐ or heterospecific) than when they were reared in isolation. D. melanogaster carrying mutations affecting short‐ or medium‐term memory showed a similar response to the social environment, not supporting a role for learning. Our results demonstrate that the amount of song a male produces during courtship is plastic depending on the social environment, which might reflect the advantage of being able to respond to variation in intrasexual competition, but that song structure itself is relatively inflexible, perhaps due to strong selection against hybridization.     

Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions. The recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.