Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol149.Issue1.2000:

Plant Science 149 1999 63 – 71 Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells Holger Spiegel a , Stefan Schillberg a,b, , Markus Sack a , Achim Holzem a , Jo¨rg Na¨hring a , Michael Monecke a , Yu-Cai Liao a , Rainer Fischer a,b a Institut fu¨r Biologie I BotanikMolekulargenetik , RWTH Aachen, Worringerweg 1 , D- 52074 Aachen, Germany b Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1 , D- 57392 Schmallenberg, Germany Received 17 February 1999; received in revised form 19 July 1999; accepted 20 July 1999 Abstract To test whether the accumulation of cytoplasmically targeted recombinant antibodies could be improved by fusion to a cytoplasmic protein, we generated a series of single chain antibody-fusion proteins and assayed the levels of functional protein. Glutathione S-transferase GST from Schistosoma japonicum, coat protein CP from TMV, thioredoxin from tobacco TRXt or thioredoxin from Escherichia coli TRXe was fused to the N-terminus of scFv24, a TMV specific single chain antibody. Accumulation of functional fusion proteins in the endoplasmic reticulum ER and plant cell cytoplasm was analysed by transient expression in tobacco leaves. ELISA analysis demonstrated that the fusion partners did not prevent the binding of scFv24 to TMV virions. However, accumulation of functional scFv24 was dependent on the fusion partner coupled to it. CP-scFv and GST-scFv fusion protein accumulation amounted to 1 mg and 3 mgg of leaf material, respectively, whereas the thioredoxin fusion proteins were produced at low levels. Western blot and surface plasmon resonance analysis confirmed the integrity of the ER retained CP and GST fusion proteins. In the cytoplasm, only the CP fusion protein was detectable 1 – 5 nggram of leaf material and levels of scFv24 alone or fused to the other three fusion partners were below the ELISA detection limit. Addition of a KDEL sequence to the C-terminus of the cytoplasmic CP fusion resulted in a 3-fold increase in protein accumulation indicating that an N-terminal CP and the C-terminal KDEL sequence are suitable elements to stabilize scFv antibodies in the cytoplasm. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Recombinant antibodies; Transgenic plants; Protein targeting; Protein stability www.elsevier.comlocateplantsci

1. Introduction

Recombinant antibody and antibody fragments rAb have been expressed in different plant cell compartments for review see [1]. The highest accumulation of functional rAb protein has been obtained in the apoplast and ER, reaching up to 6.8 of total soluble protein [2,3]. This has offered the opportunity for using plants as production vehicles for large quantities of diagnostic or thera- peutically useful antibody molecules. However, in several cases cytoplasmic expression of antibodies is desirable — to inhibit physiological functions [4 – 6] or inactivate pathogens [7 – 9]. Single chain antibodies scFv are the preferred rAb for cyto- plasmic expression since they consist of a single polypeptide that does not require in vivo assembly or complex folding [10]. Nevertheless, cytoplasmic protein levels of scFv fragments in plant cells, derived from monoclonal antibodies, are low and do not exceed 0.1 of total soluble protein [7]. It seems likely that the lack of protein disulphide isomerase and specific chaperones in the cyto- plasm results in scFv misfolding and contribute to Abbre6iations : GST, glutathione S-transferase from Schistosoma japonicum; CP, coat protein from TMV; TRXt, thioredoxin h2 from tobacco; TRXe, thioredoxin from Escherichia coli; scFv, single chain antibody; TMV, tobacco mosaic virus; N. tabacum, Nicotiana tabacum; N. benthamiana, Nicotiana benthamiana; ELISA, Enzyme- linked immunosorbent assay. Corresponding author. Tel.: + 49-241-806629; fax: + 49-241- 871062. 0168-945299 - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 4 5 - 4 enhanced proteolysis [11]. However, a recent re- port demonstrated that scFv fragments derived from phage display reached up to 0.3 and 1 of total soluble protein in the cytoplasm of Petunia leaves and petals, respectively, indicating that phage display selection can be used for enrichment of more stable scaffolds which tolerate the absence of disulfide bonds [12]. Accumulation of cytoplasmic recombinant proteins in prokaryotes has been enhanced by the addition of a stabilizing fusion protein, and anti- body stability in plants has been improved by the fusion of peptide sequences to the antibody C-ter- minal. Schouten et al. [13,14] demonstrated that addition of a C-terminal KDEL sequence signifi- cantly increased scFv protein levels in the plant cytoplasm, indicating that fusion to short polypep- tides may protect the scFv fragment from prote- olytic degradation. In Escherichia coli, linkage to N-terminal fusion partners significantly increased cytoplasmic accumulation levels of functionally active protein. Small cytoplasmic proteins, such as thioredoxin TRX, glutathione S-transferase GST or the maltose binding protein [15 – 17] were successfully used and improved the solubility and stability of heterologous proteins. The success of protein stabilization with fusion partner proteins in the E. coli system prompted us to evaluate the suitability of fusion partners to stabilize scFv fragments in the plant cytoplasm. Here, we used the TMV-specific scFv24, which has a high affinity towards epitopes present on intact virions and confers virus resistance to transgenic plants [9]. scFv24 has been successfully expressed to high level in the apoplast of tobacco plants but expression only reaches low levels in the cytoplasm [9]. Our rationale was to improve cytoplasmic protein levels by linking scFv24 to small cytoplas- mic proteins. scFv24 was fused to GST from Schistosoma japonicum, TMV coat protein CP, thioredoxin from tobacco TRXt or thioredoxin from E. coli TRXe and these scFv-fusion proteins were expressed in the ER or the cyto- plasm of tobacco leaves. The results demonstrated that all four scFv24 fusion proteins were func- tional but their protein levels were directly related to the fusion partner. The highest levels of func- tional fusion protein were observed for the ER targeted GST-scFv24 fusion protein, whereas in the cytoplasm only the CP fusion gave a signifi- cantly increased level of functional fusion protein. Cytosolic levels of CP-scFv24 could be further increased by the addition of a C-terminal KDEL sequence.

2. Materials and methods