Recombinant Hev b 1: large-scale production and immunological characterization H.-P. RIHS*, Z. CHEN*, S. SCHUMACHER*, P. ROZYNEK*, R. CREMER², M. LUNDBERG³, M. RAULF-HEIMSOTH*, A. PETERSEN§ and X. BAUR* *Research Institute for Occupational Medicine (BGFA), Bochum, Germany, ²Children's Hospital Cologne, Cologne, Germany, ³MIAB, Uppsala, Sweden, §Research Centre Borstel, Borstel, Germany Summary Background Hev b 1 represents one of the most important allergens in Hevea brasiliensis latex. It is dif®cult to get an appropriate amount of native Hev b 1 (nHev b 1) for research purposes. Objective The aim of this study was to produce suf®cient amounts of Hev b 1 by recombinant methods to prove its suitability for latex allergy diagnostics. Methods We isolated total RNA of Hevea brasiliensis leaves and synthesized cDNA by RT PCR. Recombinant Hev b 1 (rHev b 1) as well as three fragments (amino acid residues 29±137, 48±137, 78±137) were subcloned and expressed as fusion proteins with Maltose- binding protein (MBP) in Escherichia coli. The MBP-rHev b 1 fusion protein was examined by RAST with the CAP method, histamine release test and immunoblots with human sera from spina bi®da patients as well as from health care workers with latex allergy and monoclonal antibodies. Results Histamine release test and immunoblots revealed the high allergenicity of the MBP-rHev b 1 construct. By the CAP method, 54 out of 58 serum samples (93%) from latex-sensitized spina bi®da patients previously showing immunoglobulin (Ig) E to nHev b 1 exhibited IgE-binding to rHev b 1. Among 71 latex-allergic health care workers tested, 16 (22.5%) had IgE antibodies to rHev b 1. The analysis of the fusion proteins carrying rHev b 1 fragments revealed that the loss of the N-terminal 28 amino acid residues did not affect IgE-binding. In contrast, the lack of the ®rst 47 amino acid residues led to decreased IgE- binding reactivity in two out of four sera tested, whereas the absence of the N-terminal 77 residues abolished IgE-binding in these two sera. Conclusion The MBP-rHev b 1 fusion protein exhibits a corresponding IgE-binding reactivity to nHev b 1 and may therefore substitute natural Hev b 1 for both in vitro diagnostics and research purposes. Keywords: Latex allergy, Hevea brasiliensis, Hev b 1, spina bi®da, recombinant allergen Clinical and Experimental Allergy, Vol. 30, pp. 1285±1292. Submitted 30 June 1999; revised 23 September 1999; accepted 6 January 2000. Introduction The major latex allergen Hev b 1, also called rubber elongation factor, was initially isolated from latex sap of Hevea brasiliensis by Dennis and Light [1]. The enzyme prenyltransferase needs this protein to synthesize multiple cis-1,4-isopren units to rubber particles [2]. With regard to the whole protein content in latex sap, the portion of Hev b 1 reaches a value between 10 and 60% [1]. Hev b 1 is a very hydrophobic protein with a length of 137 amino acid residues and a molecular mass of 14.6 kDa. Amino acid residues like cysteine, methionine, histidine and trypto- phane are completely absent. The N-terminus of Hev b 1 includes a cluster of acidic amino acid residues and is acetylated. The function of this single post-translational modi®cation is unknown, although a protection against proteolysis is supposed [3]. In 1993, Czuppon et al. [4] Clinical and Experimental Allergy, 2000, Volume 30, pages 1285±1292 1285 q 2000 Blackwell Science Ltd Correspondence: H.-P. Rihs, BGFA, Department of Molecular Genetics, Bu Èrkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany.