Production of serpins using baculovirus expression systems Arumugam Jayakumar, a Sule Cataltepe, b YaÕan Kang, a Mitchell J. Frederick, a Kenji Mitsudo, a Ying Henderson, a Sue E. Crawford, c Gary A. Silverman, b and Gary L. Clayman a,d, * a Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Box 0441, Houston, TX 77030-4009, USA b Department of Pediatrics, Harvard Medical School, Children’s Hospital, Boston, MA 02115-5737, USA c Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA d Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Box 0441, Houston, TX 77030-4009, USA Accepted 23 June 2003 Abstract Headpin is a novel serine proteinase inhibitor (serpin) that is downregulated in many established HNSCC tumor cell lines and human oral SCC specimens. The use of the bacterial and yeast expression systems for headpin resulted in poor yields and proteins with low inhibitory activity. To circumvent these problems, we have developed a baculovirus–insect cell system for high-yield ex- pression as well as fully functional protein. Here, we describe the strategies and methods used to express headpin in an insect cell heterologous system. In addition, procedures to purify the recombinant proteins are described. A metal affinity column followed by a gel-filtration chromatography provides a rapid and efficient method for large quantity preparation of headpin. This method should be useful as an alternative expression system for those serpins that are not purifiable when expressed using the Escherichia coli or yeast expression system. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Serine proteinase inhibitor; Headpin; Recombinant baculovirus; Sf9 insect cells; Plaque assay; TALON column; Cysteine proteinases; Proteinase inhibition 1. Introduction Serpins 1 are members of a large and growing super- family of structurally related proteins displaying the capacity to form 1:1 stable complexes with proteinases [1–3]. In 1993, Remold-OÕDonnell [4] reported on a subset of serpins with a high degree of sequence simi- larity to chicken ovalbumin. Unlike the canonical a1- antitrypsin (SERPINA1), members of the ov-serpin subfamily lack both classic N-terminal signal peptides and long N- and C-terminal extensions. More than 500 serpins from a broad range of organisms including mammals, insects, certain viruses, and plants have been identified. With the possible exception of SERPINB5 (maspin), all of the 11 human ov-serpins inhibit various cysteine or serine proteinases [2]. Several members lack protease-inhibitory capability and instead have other physiological roles [5] and are known to participate in extracellular and intracellular physiological processes, including blood coagulation, inflammation, cell migra- tion, fibrinolysis, complement activation, remodeling of the extracellular matrix, hormone transport, apoptosis, cancer growth, and metastasis [2,6,7]. Heterologous expression of recombinant serpins provides unparalleled versatility for investigating the in vitro inhibitory properties and to carry out site-directed mutagenesis to study the role of specific amino acids of the reactive site loop in inhibitory activities. The use of Methods 32 (2004) 177–184 www.elsevier.com/locate/ymeth * Corresponding author. Fax: 1-713-794-4662. E-mail address: gclayman@mdanderson.org (G.L. Clayman). 1 Abbreviations used: serpin, serine proteinase inhibitor; HNSCC, head and neck squamous cell carcinoma; SCC, squamous cell carcinomas; Sf9, Spodoptera frugiperda; Sf-21, Spodoptera frugiperda; High-Five, Trichoplusia ni; FBS, fetal bovine serum; FPLC, fast- performance liquid chromatography; SEC, size exclusion column; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electropho- resis; P1, passage 1; P2, passage 2; P3, passage 3; mAb, monoclonal antibody; M r , average molecular weight. 1046-2023/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1046-2023(03)00209-3