IN VIVO AND IN VITRO PRODUCTION OF mAbs 599 Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T. & Winter, G. (1989), Binding activities of a repertoir of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 341, 544-546. Ward, M., Wilson, L.J., Kodama, K.H., Rey, M.W. & Berka, R.M. (1990), Improved production of chymo- sin in Aspergillus by expression as a glucoamylase- chymosin fusion. Bioflechnology, 8, 435-440. Wu, X.C., Ng, S.C., Near, R.I. & Wong, S.L. (1993) Effi- cient production of a functional single-chain antidi- goxin antibody via an engineered Bacillus subtilis expression-secretion system. Bio/Technnology, 11, 71-76. Yu, H., Nakano, Y., Yamashita, Y., Oho, T. & Koga, T. (1997) Effects of antibodies against cell surface pro- tein antigenPAc-glucosyltransferase fusion proteins on glucansynthesis andcell adhesion of Streptococ- cusmutans. Infect. Immun., 65, 2292-2298. In vitro production of recombinant antibody fragments in Pichiu pastoris C.A. Pennell cl)(*) and P. Eldin c2) (‘) Department of Laboratory Medicine and Pathology & University of Minnesota Cancer Center, University of Minnesota, Minneapolis, MN 55455 (USA), and (2) Dynamique Moleculaire des Interactions membranaires, Montpellier, Cedex 5 (France) Introduction The methyltropic yeast Pichia pastoris is rapidly becoming a preferred host for the efficient expres- sion of heterologous proteins (reviewed in Hollen- berg and Gellissen, 1997 ; Sreekrishna et al., 1997 ; Sudbery, 1996). P. pastoris combines the general features of protein expression in eukaryotes with the fast growth and genetic modifiability of prokaryotes. It is less expensive than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture, and it typically yields higher quantities of secreted functional proteins. The ability to correctly express folded secreted proteins, including highly disulphide-bonded ones (White et al., 1994), pro- vides a distinct advantage over bacterial systemsthat often require laborious and inefficient procedures to denature and refold proteins expressed as insoluble, inclusion bodies (Skerra, 1993). P. pastoris is a superior expression host over its more famous non- methyltropic relative, Saccharomyces cerevisiae, because P. pastoris grows stably to high cell den- sities in fermentors (> 100 mg dry weight per ml cul- ture) and it has strong, tightly regulated promoters (Ellis et al., 1985). For thesereasons,P. pastoris has recently been exploited as an expression system for the high-level secretion of many proteins, including recombinant antibody fragments. By definition, methyltropic yeasts are capable of utilizing methanol as their sole carbon source. The first enzyme in the methanol-utilization pathway, alcohol oxidase (AOX), is encoded by two closely related genes: AOXI and AOX (Ellis et al., 1985; Cregg et al., 1989). Although the AOXl and AOX proteins have 97 % sequenceidentity and equivalent enzymatic activity, over 95 % of the alcohol oxidase activity in P. pastoris is attributable to AOXl. This is due to the strength of the AOXI promoter (Cregg et al., 1989; Koutz et al., 1989). Upon the addition of methanol, AOXI gene transcription is rapidly induced to high levels and ultimately accounts for 5% of the total polyA+ RNA (Cregg and Madden, 1988). The AOXl protein is correspondingly over- expressed and comprises up to 30% of the total intracellular protein. Heterologous genes are there- fore cloned under control of the AOXZ promoter to ReceivedJuly 10, 1998. (*) To whom correspondence should be addressed.