High-level secretion of two antibody single chain Fv fragments by Pichia pastoris
Introduction
Antibody single chain Fv (sFv) fragments are potentially more effective than whole antibodies for the diagnosis or therapy of many diseases (Huston et al., 1993). sFv fragments contain heavy and light chain variable (VH and VL) regions connected by a small, flexible peptide and as such, are about a fifth the size of intact IgG (Bird et al., 1988; Huston et al., 1988). The reduced size of sFv fragments (27 000–30 000 Mr) permits them to penetrate tissues and solid tumors more readily than whole antibodies, and to be cleared more rapidly from the blood (Yokota et al., 1992). These properties make sFv fragments and their derivatives well suited for in vivo applications such as the treatment of drug overdoses, medical imaging, and tumor targeting (Huston et al., 1993; Yokota et al., 1992; Pastan et al., 1995). However, the in vivo use of sFv fragments often requires large (milligram to gram) quantities of protein that can be difficult or expensive to obtain with current sFv expression systems (Skerra, 1993; Kitchin et al., 1995). To avoid these difficulties, we have explored using the methylotrophic yeast Pichia pastoris to express potentially therapeutic sFv fragments.
P. pastoris combines the general features of eukaryotic protein expression with fast growth (Cregg and Higgins, 1995). Heterologous proteins can be expressed intracellularly or secreted when linked to the appropriate signal sequences. Although their secretion of endogenous proteins is very low, P. pastoris cells have the capacity to secrete grams per liter of foreign proteins in a protein-poor medium that is inexpensive and chemically defined (Laroche et al., 1994; Cregg and Higgins, 1995). The low level of protein in the medium formulation facilitates purification of the final secreted product.
Protein expression in P. pastoris is based on the use of the alcohol oxidase gene, AOX1 (Cregg and Higgins, 1995). Transcription of heterologous genes is tightly regulated by the AOX1 promoter when the AOX1 coding sequence is replaced by a gene of interest. Genes under control of the AOX1 promoter are rapidly transcribed upon addition of methanol, the substrate of alcohol oxidase. The utility of this system for our purposes is suggested by the expression of several mammalian proteins with therapeutic value such as vascular endothelial growth factor, tumor necrosis factor, and the anticoagulant-antimetastatic protein ghilanten (Mohanraj et al., 1995; Sreekrishna et al., 1989; Brankamp et al., 1995).
Here we report the secretion of two murine sFv fragments by P. pastoris. The first sFv fragment is specific for desipramine (DMI), a tricyclic antidepressant drug (Litovitz et al., 1995). DMI-specific sFv fragments show promise in the treatment of DMI overdoses because they induce a rapid and marked redistribution of subtoxic, tracer doses of 3H-DMI from tissues into the sera of rats (Shelver et al., 1996). This rat model is of clinical interest because tricyclic antidepressant toxicity is the leading cause of death from intentional drug overdose in the United States (Litovitz et al., 1995). The second sFv fragment is specific for CD7, a 40 000 Mr transmembrane glycoprotein expressed at high densities on almost all human T cell acute lymphoblastic leukemias (T-ALL) but not on the vast majority of normal cells (Barcena et al., 1995). Its tissue distribution pattern makes CD7 an excellent molecule to target for T-ALL therapy (Amlot and Cammisuli, 1990). To produce sufficient amounts of sFv protein for therapy in a mouse model of human T-ALL (Jansen et al., 1992), and for the treatment of rats given toxic (rather than tracer) doses of DMI, the anti-CD7 and anti-DMI sFv fragments have now been expressed in P. pastoris. Below we describe the expression and functional characterization of both proteins.
Section snippets
sFv cloning
sFv fragments were derived from murine hybridomas designated 3A1f (anti-human CD7) (Palker et al., 1985) and G5 (anti-DMI) (Bowles et al., 1988) using reverse-transcription PCR followed by splice overlap extension (Ho et al., 1989). The 3A1f sFv construct was then modified by PCR to include a C-terminal cysteine codon to facilitate linking the expressed sFv fragment (3A1f sFvcys) to other proteins. The construction of these sFv fragments were detailed elsewhere (manuscripts submitted). Both sFv
Cloning and expression of sFv genes
The anti-CD7 and anti-DMI sFv fragments were derived from the murine hybridomas 3A1f (Palker et al., 1985) and G5 (Bowles et al., 1988), respectively. The 3A1f sFv fragment was engineered to contain a C-terminal cysteine (3A1fcys) to facilitate its conjugation to toxins. Both proteins were initially expressed in E. coli but their yields were limited by either low level production (0.25 mg/l) of soluble 3A1fcys sFv protein or by the inefficiency inherent to refolding denatured inclusion bodies
Conclusions
Our studies highlight the utility of expressing sFv fragments in P. pastoris. We have shown that: (1) sFv fragments can be secreted at high levels (up to 250 mg/l for G5); (2) the soluble sFv fragments are easily and efficiently purified in one step from the culture supernatants; and (3) the soluble fragments are indistinguishable from their counterparts produced in bacteria in terms of affinity. Recently, other sFv fragments have been expressed in P. pastoris but at yields lower than 250 mg/l (
Acknowledgements
We thank Drs. Barton Haynes (Duke University), Tucker LeBien (University of Minnesota), and Ray Mernaugh (Pharmacia Biotech) for kindly providing the 3A1f hybridoma, the biotinylated antibodies, and the anti-E tag affinity column, respectively. G.L. was supported by a National Institute of General Medical Sciences Biotechnology Training Fellowship (IT32-GM08347). P.E. was sponsored by a postdoctoral fellowship from Elf Aquitaine, Inc. (France) while M.E.P. was supported by a predoctoral
References (27)
- Amlot, P. and Cammisuli, S. (1990) CD7 monoclonal antibodies. In: C.A.K. Borrebaeck and J.W. Larrick, (Eds.)...
- Anna-Arriola, S.S. and Herskowitz, I. (1994) Isolation and DNA sequence of the STE13 gene encoding dipeptidyl...
- Barcena, A., Muench, M.O., Roncarolo, M.G. and Spits, H. (1995) Tracing the expression of CD7 and other antigens during...
- Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M., Lee, T., Pope, H.S., Riordan, G.S....
- Bowles, M., Johnston, S.C., Schoof, D.D., Pentel, P.R. and Pond, S.M. (1988) Large scale production and purification of...
- Brankamp, R.G., Sreekrishna, K., Smith, P.L., Blankenship, D.T. and Cardin, A.D. (1995) Expression of a synthetic gene...
- Cregg, J.M. and Higgins, D.R. (1995) Production of foreign proteins in the yeast Pichia pastoris. Can. J. Botany 73,...
- Damle, N.K. and Aruffo, A. (1991) Vascular cell adhesion molecule 1 induces T-cell antigen receptor-dependent...
- Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R. (1989) Site-direct mutagenesis by overlap extension...
- Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.-S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri,...
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