SummaryNucleotide sequences homologous to arpA encoding the A-factor receptor protein (ArpA) of Streptomyces griseus are distributed in a wide variety of streptomycetes. Two genes, cprA and cprB, each encoding an ArpA-like protein were found and cloned from Streptomyces coelicolor A3(2). CprA and CprB shared 90.7% identity in amino acid sequence and both showed about 35% identity to ArpA. Disruption of cprA by use of an M13 phage-derived single-stranded vector resulted in severe reduction of actinorhodin and undecylprodigiosin production. In addition, the timing of sporulation in the cprA disruptants was delayed by 1 day. The cprA gene thus appeared to act as a positive regulator or an accelerator for secondary metabolite formation and sporulation. Consistent with this idea, introduction of cprA on a low-copy-number plasmid into the parental strain led to overproduction of these secondary metabolites and accelerated the timing of sporulation. On the other hand, cprB disruption resulted in precocious and overproduction of actinorhodin. However, almost no effect on undecylprodigiosin was detected in the cprB disruptants. Sporulation of the cprB disruptant began 1 day earlier than the parental strain. The cprB gene thus behaved as a negative regulator on actinorhodin production and sporulation. Consistent with this, extra copies of cprB in the parental strain caused reduced production of actinorhodin and delay in sporulation. It is thus concluded that both cprA and cprB play regulatory roles in both secondary metabolism and morphogenesis in S. coelicolor A3(2), just as the arpA /A-factor system in Streptomyces griseus.

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When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.Antibodies for pharmaceutical applications have recently received a great deal of attention due to their specific antigenbinding activities, antibody-dependent cellular cytotoxicity, and other useful characteristics. Indeed, sales of antibodies for pharmaceuticals have grown rapidly over the past decade and are expected to exceed 30 billion U.S. dollars by 2010 (1). A large volume of antibodies is required for pharmaceuticals, as antibodies are primarily marketed for chronic conditions. Antibodies possess relatively low potency, which results in the need for accumulating doses over time (10). Although therapeutic antibodies have great potential value, they have also been viewed with some skepticism. Antibodies are expensive to produce, in part because they are commonly manufactured by using batch/fed-batch cultures of mammalian cells. The increasing demand to reduce the cost of antibody production has promoted research into the development of an antibody expression system that is more practical than expression with mammalian cells.Development of transgenic plants and animals as alternative hosts is therefore a promising field of study. Monoclonal antibodies have, thus far, been successfully produced from a number of sources, including plants, the milk of transgenic goats, the eggs of transgenic chickens, etc. (4, 12, 32). Moreover, antibodies derived from certain newly developed transgenic systems share physical characteristics that are similar to those of antibodies from mammalian cells such as Chinese hamster ovary (CHO) cells while exhibiting higher antibody-dependent cellular cytotoxicity activity due to the absence of fucose residues in N-linked sugar chains (6, 45). These alternative transgenic expression systems could reduce the cost of large-scale antibody production. However, the prolonged construction of transgenics remains a major disadvantage in terms of market demands.The production of antibodies and antibody fragments has been studied by using various microorganism expression systems, including Escherichia coli, fungus, and yeast (7,13,37,41). Since microorg...

The aim of this study was to classify the retention time of recombinant human monoclonal antibodies (rhMAbs) in hydroxyapatite chromatography with sodium-phosphate gradient elution according to their physicochemical properties. We analyzed 37 rhMAbs and found that (i) the structures of both the constant and variable regions affected retention time, (ii) the number of basic amino acid residues in the variable region, particularly in the heavy chain, correlated well with retention time, and (iii) this correlation was more pronounced (e.g. r(2)=0.87 for 18 kappa IgG(1) rhMAbs) when the surface accessibility of those residues are taken into account. These findings provide a useful guide for investigators and purification-process developers working with monoclonal antibodies.