Titer estimation for quality control (TEQC) method: a practical approach for optimal production of protein complexes using the baculovirus expression vector system

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain the protein or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the ‘initial infectivity’ was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

Inter-specific parasite competition: mixed infections ofSchistosoma mansoniandS. rodhainiin the definitive host

SUMMARYCompetition between parasite species has been predicted to be an important force shaping parasite and host ecology and evolution, although empirical data are often lacking. Using theMus musculus-Schistosoma mansoniandSchistosoma rodhainihost-parasite systems we characterized mate choice and inter-specific competition between these two schistosome species. Simultaneous infections revealed species-specific mate preferences for both species as well as suggesting mating competition, with maleS. rodhainiappearing dominant over maleS. mansoni.S. rodhainihomologous pairs were also shown to have increased reproduction per paired female in the presence of a competitor in simultaneous infections. Overall total reproductive success was, however, similar between the two species under conditions of direct competition due to the greater initial infectivity ofS. mansoniin comparison toS. rodhaini. Inter-specific competition was also implicated as increased parasite virulence to the host. The potential effects of such interactions on parasite and host ecology and evolution in nature are discussed.

Ultracentrifugal studies of the infective and complement-fixing components in the virus system of foot-and-mouth disease

The sedimentation constant of the infective particle in foot-and-mouth disease has been determined by modifications of the methods of Elford (1936) and Polsen (1941). A new high-speed, swinging-cup rotor was employed. A sedimentation constant of 70 Svedberg units was obtained for the infective particle in a variety of starting materials derived from guineapigs, mice and cattle. The validity of the data is discussed in relation to the accuracy with which a sedimentation constant may be determined by these methods. Ultracentrifugal studies employing inclined tubes have demonstrated that in fresh preparations the infective particle is associated with from 0 to 50% of the initial complement-fixing activity. The remaining complement-fixing activity is associated with a component of sedimentation constant 8 Svedberg units. This slower sedimenting component, if infective, contributes less than 0⋅01% of the initial infectivity. A direct and relatively precise method is described for the determination of the partition of a biological activity between two or more components of a virus system. By the use of radial and inclined tubes in non-optical procedures a correlation between these methods has been established. It is shown that the sedimentation constant of a biologically active component may be estimated by procedures based on sampling in inclined tubes. The G integral is introduced as an accurate and convenient parameter which greatly facilitates the calculation and presentation of the results of ultracentrifugal studies.