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Baculovirus expression systems for recombinant proteins have found widespread use because they produce biologically active proteins more reliably than do bacterial or yeast expression systems, and the yields generally exceed those from mammalian expression systems. However, optimization of baculovirus yields remains an important issue, especially when biophysical studies are contemplated. Recently we sought to produce the recombinant cysteine proteases, cathepsins B and S (1,4,16), from baculovirus for structural studies. Here, we report that production of these recombinant cathepsins reached the high levels we needed for this objective when the cultures were inoculated at multiplicities of infection (MOIs) several orders of magnitude lower than those conventionally recommended. For baculoviral expression of preprocathepsins B and S, the respective cDNAs were cloned from human liver cDNA by reverse transcription polymerase chain reaction (RT-PCR) using primers designed according to published nucleotide sequences (4,16). The cDNA encoding the 30-kDa fragment of fibronectin (FN-30) (5,8) was obtained by RT-PCR of total RNA from human dermal fibroblasts, again using primers corresponding to the published cDNA sequence. These cDNA inserts, which each contained endogeneous signal sequences for secretion, were separately subcloned into the pFASTBAC 1 donor plasmid (Life Technologies, Gaithersburg, MD, USA). A bacterial strain (DH10B) containing a vendorsupplied baculovirus shuttle vector (bacmid; Life Technologies) was transformed with recombinant pFASTBAC 1 plasmid, and colonies containing recombinant bacmids were grown for isolation of the resulting viral DNA. Transfections of Sf9 Insect Cells (PharMingen, San Diego, CA, USA) in log-phase growth were performed using CELLFECTIN Reagent (Life Technologies). Supernatants were used to infect Sf9 cells through two or three stages of amplification, initially in monolayer cultures, then at the final stage in shaker flasks. High viral titers (ca. 1–3 × 108 ) were obtained after final amplification, as indicated by plaque assays and end-point dilution assays. At all times during amplification and subsequent culture, insect cells were maintained at 27°–28°C. Following inoculation of 1 × 106 Sf9 cells/mL in Grace’s insect cell media containing 10% fetal bovine serum (FBS) (the presence of which facilitated initial infection), 0.1% pluronic acid, antibiotics and amphotericin (Life Technologies), cells were grown in shaker culture at 125 or 250 mL/flask. At 24 h after inoculation, cells were centrifuged, resuspended in Sf-900II Serum-Free Media (Life Technologies) supplemented with 1 mL of 10% pluronic acid per 100 mL media (which we had found to increase yields approximately twofold) and cultured as before. This transfer to

When Less is More: Enhanced Baculovirus Production of Recombinant Proteins at Very Low Multiplicities of Infection