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Biotechnology and Applied Biochemistry (2003) 38, (25–33) (Printed in Great Britain)

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Expression of the Bacillus thuringiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct

Cemal Gurkan¹ and David J. Ellar

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.

Key words: Bacillus thuringiensis, cytolytic/haemolytic/insecticidal toxin, membrane-acting immunotoxin, recursive PCR, subspecies kyushuensis, total gene synthesis.

Abbreviations used: IT, immunotoxin; syncyt2Aa1, synthetic cyt2Aa1 gene; Mut^s, methanol utilization slow; rPCR, recursive PCR; oePCR, overlap extension PCR; M.I., methanol induction.

¹To whom correspondence should be addressed, at (present address) Department of Cell Biology, MB6, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, U.S.A. (e-mail cemalgurkan@cal.berkeley.edu).

The nucleotide sequence data corresponding to the syncyt2Aa1 open reading frame was deposited with the EMBL/GenBank® Nucleotide Sequence Databases under the accession number AF398463.

Bacillus thuringiensis d-endotoxins are membrane-active, pore-forming proteins with highly specific insecticidal activities. In addition to a well-established role in the biological control of a wide variety of crop pests and disease vectors, these toxins also have great potential for the development of anti-tumour agents called immunotoxins (ITs), chimaeric molecules consisting of a cell-binding ligand coupled to a toxin or its subunits. The ultimate goal of our study was the recombinant production of such ITs based on the Cyt2Aa1 toxin from B. thuringiensis subspecies kyushuensis. We explored the use of Pichia pastoris for recombinant IT production because earlier attempts in our laboratory using the Escherichia coli expression system or various chemical conjugation strategies yielded only low levels of functional product. However, our initial attempts were not successful because the A+T-rich bacterial cyt2Aa1 gene contained fortuitous polyadenylation sites, causing premature transcription termination in this yeast. Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1) optimized for heterologous expression in P. pastoris. This was achieved by increasing the overall G+C content of the bacterial cyt2Aa1 while changing its codon usage to that preferred by the methylotrophic yeast. Here we describe in detail the design, synthesis and requisite PCR repair of syncyt2Aa1, then present analyses of recombinant Cyt2Aa1 expression in P. pastoris using this synthetic gene. Following the results presented in this paper, the syncyt2Aa1 gene was also successfully used for the recombinant production of a Cyt2Aa1-based IT in the same expression host [C. Gurkan and D. J. Ellar (2003) Protein Expression Purification 29, 103–116].

Received 27 January 2003/6 March 2003; accepted 11 March 2003

Published as Immediate Publication 11 March 2003, DOI 10.1042/BA20030017

© 2003 Portland Press Ltd