Fusion of Tat-PTD to the C-terminus of catfish growth hormone enhances its cell uptakes and growth-promoting effects Jingyou Yu a , Xiaolin Meng a, ⁎, Jinping Xu a, ⁎, Dandan Chen a , Mingxiang Meng b , Yawen Ni a a State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, 430072, PR China b Department of Urology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China abstract article info Article history: Received 22 June 2012 Received in revised form 15 January 2013 Accepted 3 February 2013 Available online 12 February 2013 Keywords: Silurus asotus GH Transduction Growth promotion Common carp Tat Fish growth hormone has been long regarded as a promising feed additive in aquaculture for its significant growth promotion effect. To improve its efficiency in oral applications, we generated a Silurus asotus growth hormone-Tat (saGH-Tat) fusion protein and investigated its effect on biological activities relevant to transduc- tion and growth promotion. In this study, saGH, saGH-Tat, Green fluorescent protein and saGH fused protein (GFP-saGH) and GFP-saGH-Tat were all highly expressed in Escherichia coli strain BL21 (DE3) and confirmed by Western blot. The transduction effect of Tat protein transduction domain (Tat-PTD) was verified by observ- ing increased GFP fluorescence in separated intestine cells or frozen sections in GFP-saGH-Tat treated fish. The recombinant saGH and saGH-Tat were tested for biological activity by specific binding with crude liver membrane extracts as well as in a growth promotion assay via oral administration. saGH-Tat treatment dem- onstrated strengthened receptor binding and accelerated growth-promoting effects. Under optimal concen- trations of hormone (1 μg/g of fish body weight), saGH-Tat treated fish exhibited an 87.25% growth increase compared to 61.3% in saGH-treated fish. The response of downstream signal pathway factors were analyzed by measuring insulin like growth factor-I (IGF-I), growth hormone receptor (GHR) mRNA expression in the liver, and growth related digestive enzymes (trypsin and chymotrypsin), and all indicate significant up-regulation. Further confirmation of increased GH level in carp blood by Tat fusion was shown by flow cy- tometry. These results suggested that Tat-PTD fused to the C-terminus saGH greatly enhances its biological ac- tivities, making it a promising agent for increasing productivity in aquaculture. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Fish growth hormone (fGH) is an approximately 22 kDa single- chain polypeptide produced by anterior pituitary cells that is involved in the regulation of growth and development (Biga and Meyer, 2009; Perez-Sanchez and Le Bail, 1999), appetite, metabolism, reproduction (Gac et al., 1993), immunomodulation (Yada, 2007) and osmoregula- tion (McCormick, 2001; Sakamoto et al., 1993). Since the demonstra- tion that recombinant fish GH has the same functions as native GH, expression of fish GH in a series of hosts was explored including Escherichia coli (Sekine et al., 1985), yeast Picha pastoris (Li et al., 2003) and various other expression systems (Kawata et al., 1991; Kim et al., 2002). Multiple delivery routes for recombinant GH to fish have also been developed, such as injection, oral administration, immersion treat- ment and others (Crim, 1985). However, among these delivery routes, oral administration appears to be the most practical method to date because it does not require the fish farmer to handle each fish individ- ually (Jeh et al., 1998). The main challenge for large scale application of recombinant GH is its limited utilization efficiency (Ben-Atia et al., 1999; Hertz et al., 1991), which may cause significant protein loss leading to high production costs. Several attempts have been made to enhance intestinal absorption of recombinant GH, such as mixing with a protective agent like the detergent deoxycholate (Hertz et al., 1991), slow-releasing formulations (Leedom et al., 2002a,b) or enteric polymer matrices (Moriyama et al., 1993; Promdonkoy et al., 2004). However, these methods add either extra materials or uncertain food-safety risks. Thus, a more economical and safer route is needed. Cell penetrating peptides (CPPs) present one potential solution. Numerous studies have reported that carrier peptides can deliver a wide range of cargoes to a diverse range of cells and tissues (Dietz and Bohr, 2004; Simon et al., 2011). Tat (transactivator of transcrip- tion), one of the most extensively studied CPPs, has been previously reported to mediate internalization of heterogonous protein into liv- ing cells (Fawell et al., 1994). Tat protein transduction domains (Tat-PTDs) refer to the transduction domain derived from the HIV Tat protein which is an 86 amino acid protein involved in the replica- tion, neuro-toxicity, and immune activation of HIV-1. Despite its length, however, only a short cluster of basic amino acids is necessary Aquaculture 392–395 (2013) 84–93 ⁎ Corresponding authors. Tel./fax: +86 27 68754217. E-mail addresses: jyu@whu.edu.cn (J. Yu), mengxiaolin8@hotmail.com (X. Meng), jpxu@whu.edu.cn (J. Xu). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.02.003 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online