Nucleic Acids Research, 2005, Vol. 33, No. 12 e109 doi:10.1093/nar/gni103 A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system Yasuhiro Suzuki, Naoko Kagawa1,3, Toru Fujino1,3, Tsuyoshi Sumiya 3 , Taichi Andoh1,3, Kumiko Ishikawa1,3, Rie Kimura3, Kiyokazu Kemmochi2, Tsutomu Ohta4 and Shigeo Tanaka1,3,* Primer Production Department, 1Research and Development Department and 2Service Laboratory Department, Invitrogen Corporation,Yokohama Kanazawa High-Tech Center, Techno-Core Building.4F 1-1-1 Fukuura, Kanazawa, Yokohama, Kanagawa, 236-0004 Japan, 3Japan Biological Information Consortium, Grande Building5F, 2-26-9 Hacchoubori Chuoh Tokyo, 104-0032, Japan and 4Center for Medical Genomics, National Cancer Center Research Institute 5-1-1 Tsukiji, Chuoh, Tokyo, 104-0045, Japan Received April 27, 2005; Revised and Accepted June 10, 2005 ABSTRACT There is an increasing demand for easy, high- throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have char- acterized this novel HTP method on 96 targets with .90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors. INTRODUCTION As structural and in silico biology have advanced, systematic and robust high-throughput (HTP) methods for protein engin- eering are required to generate mutants that will allow us to model and analyze protein function. Production of protein drugs, including chimeric antibodies, requires a method for mutagenesis and chimeragenesis. While many methods for chimeragenesis, such as domain shufﬂing (1,2), have been developed, the majority utilize complex processes to obtain the desired protein and are not amenable to HTP automation. Recently, the cloning systems with in vitro recombination were developed (3,4) and are amenable to HTP cloning. Gateway cloning technology, one of such systems, utilizes the bacteriophage l recombination system in vitro (4). We characterized this recombination system and succeeded in introducing N- and C-terminal fragments on each recombina- tion site by in vitro recombination and cloning in an HTP manner. This cloning strategy represents an easy HTP method for site-directed chimeragenesis, protein engineering and mutagenesis. In the Gateway cloning system, a fragment carrying attB1 and attB2 recombination sites on both ends is prepared by PCR and introduced into a Donor vector carrying attP1 and attP2 recombination sites by an in vitro recombination reaction driven by BP clonase. attB1, attB2, attP1 and attP2 sequences are designed from natural attB and attP sequences, respect- ively, to obtain speciﬁcity without reduced recombination efﬁ- ciency. There are two processes in Gateway cloning: the ﬁrst is intermolecular recombination between a fragment and a plas- mid at attB1 and attP1 or attB2 and attP2 and the second is intramolecular recombination. We have developed a novel strategy for Gateway cloning, named ‘N- and C-term’ (N- and C-terminal) Gateway cloning, in which two independ- ent fragments, each carrying attB1 or attB2 on their ends, are combined with a single plasmid containing both attP1 and attP2 sites. The resulting linear constructs carrying both N- and C-terminal fragments are circularized and the Gateway clone is constructed. It was reported that homologous recomb- ination in Escherichia coli was available for DNA cloning (5,6). We adopted homologous recombination in E.coli to circularize a plasmid. This cloning technique offers a rapid, automatable way to construct chimeric genes and introduce *To whom correspondence should be addressed. Email: shigeo.tanaka@invitrogen.com The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org