Biotechnology Letters 26: 1247–1252, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 1247 Cloning and characterization of the bovine testicular PH-20 hyaluronidase core domain Srimek Chowpongpang 1,∗ , Haw-Shook Shin 2 & Eun-Ki Kim 2 1 National Center for Genetic Engineering and Biotechnology, Plant Genetic Engineering Unit, Kasetsart Univer- sity, Khamphaengsaen, Nakhon Pathom 73140, Thailand 2 Department of Biological Engineering, Inha University, Incheon 402-751, South Korea ∗ Author for correspondence (Fax: +66-34-351-908; E-mail: srimake@biotec.or.th) Received 19 March 2004; Revisions requested 7 April 2004; Revisions received 2 June 2004; Accepted 2 June 2004 Key words: homology sequences, hyaluronan, Pichia pastoris, Spam1 Abstract The core nucleotide sequence of bovine (Bos taurus) testicular PH-20 hyaluronidase was cloned using one step RT- PCR. The 5 ′ and 3 ′ regions were cloned separately and a sequence overlap of 124 bp facilitated the fusion of these two fragments by overlapping PCR, resulting in a concatenated sequence of 1422 bp. This nucleotide sequence and its deduced amino acid sequence were compared to homologous sequences from eight other mammal species. The bovine sequences were most similar to those of the pig, Sus scrofa (swine Spam1: 79.1% nucleotide and 70.1% amino acid similarity) and least similar to sequences from the Norway rat, Rattus norvegicus (murine Spam1: 61% nucleotide and 53.3% amino acid similarity). A phylogenetic analysis joined the red fox (Vulpes vulpes) sequence as sister to the bull-pig pair. Twelve cysteine residues were conserved among all nine aligned amino acid sequences and five proposed glycosylation sites have been identified. The feasibility of developing an effective, low-cost bovine PH-20 expression system is discussed in light of these new data. Introduction Most vertebrate connective tissue contains hyalur- onan, a simple glycosaminoglycan that can be hy- drolyzed by a hyaluronidase (Gmachl & Kreil 1993, Hynes & Walton 2000, Zhang & Martin-DeLeon 2001). PH-20 is a testes-specific hyaluronidase that is bound to the acrosomal membrane of the sperm head (Gmachl et al. 1993, Hunnicutt et al. 1996) and plays a role in fertilization. PH-20 cDNAs from humans and a variety of other mammals have been cloned, char- acterized and expressed in mammalian cell lines (ten Have et al. 1998, Zhang & Martin-DeLeon 2001). In addition, PH-20 active sites have been mapped in hu- mans and crab-eating macaques (Macaca fasciculata). These studies reveal that the glycosylphosphatidyl- anchored domain is on the carboxyl terminus (Cherr et al. 2001) and that glycosylation occurs during mat- uration (Li et al. 2002). Post-translational proteolytic processing, including disulfide bond formation and modification by a signal peptide, are necessary for the enzyme to attain full activity (Meyer et al. 1997, Li et al. 2002). The ability of PH-20 to depolymerize the hyalur- onan in connective tissue has been put to a variety of uses, including cancer diagnosis (Godin et al. 2000) and inhibition of malignant tumor growth. Topical ap- plication can alleviate joint problems, soften dry skin, loosen the foreskin of uncircumcised men and accel- erate wound healing after optical surgery (West et al. 1997). This molecule has a number of interesting features and promising applications but a complete understand- ing of post-translational processing is necessary to gain maximal activity in alternate expression systems. Herein we describe our methods for successfully clon- ing and characterizing the core nucleotide sequence of bovine testicular PH-20 sequence and its deduced amino acid sequence (GenBank accession number AY297029). We discuss the structure of this gene re-