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DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E o ‫؍‬ ؊122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys 30 -Pro 31 -His 32 -Cys 33 ) is primarily due to an extremely low pK a value (3.4) of Cys 30 , which is stabilized by the partial positive dipole charge of the active-site helix ␣1 (residues 30 -37). We have randomized all non-cysteine residues of helix ␣1 (residues 31, 32, and 34 -37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines ␣-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below ؊210 mV. All inactive variants had redox potentials lower than ؊210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.

Recombinant baculoviruses derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) are widely used to express heterologous genes in insect cells, but the use of the baculovirus expression vector system (BEVS) is hampered by slow and tedious procedures for the selection and separation of baculovirus-infected insect cells and for titer determination. Here we developed a new technology based on the bicistronic vector with a fusion protein of the human integral plasma membrane glycoprotein CD4 and green fluorescent protein (GFP) for concomitant expression of target proteins in insect Sf21 cells. Magnetic cell sorting (MACS) technology with anti-CD4 antibody-labeled superparamagnetic beads was used to separate the baculovirus-infected from the noninfected insect cells and therefore to increase the virus titer and to reduce process time. With the herein described use of the MACS-improved baculovirus expression plasmid MACS in baculovirus expression (pMACS-iBac-1), we have been able to select the baculovirus-infected insect cells at an early time point of the infection cycle and therefore enrich the virus titer dramatically. Furthermore, simple end point dilution and GFP fluorescence detection can be used for early and facile detection of recombinant viruses and simplified titer determinations. We show that the bicistronic pMACSiBac-1 with an additional multiple cloning site under the control of the very late promoter polyhedrin (PPH) allows for the expression of target proteins in high amounts, less workloads, and shorter timelines.