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A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Proteinbased microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.

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NAD+ -dependent formate dehydrogenase (FDH; EC 1.2.1.2) plays an important role in methylotrophic yeasts, catalysing the final step in the methanol oxidation pathway [1,2]. In general, FDH is a highly conservative enzyme. The primary sequence homology is no < 80-85% between enzymes of the same organismal group and 50-55% or more between enzymes from different groups. In addition, the catalytic amino acids, as well as the amino acids that contribute to the structural stability, are almost totally conserved (sequence homology of approximately 95%). Formate dehydrogenase from Candida boidinii (CboFDH) is a dimer with two identical subunits, each possessing an independent active centre [1,3].Detailed studies of FDH are justified by the considerable biotechnological potential of this enzyme. For example, it is used as a diagnostic enzyme for the determination of formic acid [4] . Based on structural information for CboFDH, the loop region between b-sheet 7 and a-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues (Asp195 and Tyr196) that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site-saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln ⁄ Tyr196His, which showed a more than 2 · 10 7 -fold improvement in overall catalytic efficiency with NADP + and a more than 900-fold decrease in the efficiency with NAD + as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible for coenzyme specificity. The double mutant Asp195Gln ⁄ Tyr196His was tested for practical applicability in a cofactor recycling system composed of cytochrome P450 monooxygenase from Bacillus subtilis, (CYP102A2), NADP + , formic acid and x-(p-nitrophenyl)dodecanoic acid (12-pNCA). Using a 1250-fold excess of 12-pNCA over NADP + the first order rate constant was determined to be equal to k obs = 0.059 ± 0.004 min )1 .Abbreviations 12-pNCA, x-(p-nitrophenyl)dodecanoic acid; CboFDH, Candida boidinii FDH; CYP102A2, cytochrome P450 monooxygenase from Bacillus subtilis; FDH, formate dehydrogenase; G6PDH, glucose 6-phosphate dehydrogenase; PTDH, phosphite dehydrogenase.

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