Preparation of recombinant human canstatin using transgenic <italic>Dunaliella salina</italic>

Abstract

Canstatin, which possesses a significant inhibition effect on the migration of endothelial cells and a strong anticancer effect [1,2], has been applied in the treatment of many cancers including human oral, breast, prostate, pancreatic, and colorectal [3–7]. However, because the expression of bioactive recombinant canstatin is very low using the current expression systems, e.g. prokaryotic Escherichia coli expression system [6], its application has still been limited to clinical trials. Several eukaryotic cell expression systems have been exploited for canstatin production, such as Bombyx mori cells [8] and Drosophila melanogaster S2 cells [9], but they also have a lot of disadvantages, for example, high culture cost, poor yield, and difficulty in purification. Therefore, it is necessary and urgent to develop an optimal expression system for the large-scale production of the recombinant canstatin. Transgenic Dunaliella salina (UTEX-1644) system as a novel potential bioreactor [10,11] is an optimal alternative for the production of the recombinant canstatin due to the following advantages: (i) this expression system has the potential for large-scale production of exogenous proteins; (ii) D. salina has been extensively used in industrial and pharmaceutical areas owing to the capability of accumulating valuable fine components such as carotenoids, vitamins, minerals, and proteins; (iii) D. salina cells themselves are natural protoplasts, and can be easily transformed and cultured; (iv) eukaryotic D. salina cells have the post-transcriptional and post-translational modifications for the production of bioactive proteins. In the present study, therefore, we tried to develop a novel eukaryotic expression system for the recombinant canstatin by using transgenic D. salina, and this system will provide a new, safe, and environmental protection platform for the large-scale production of human recombinant canstatin. The main materials used in this study were as follows: plasmids pBI221-bar and pUV-GUS were obtained as a gift from Prof. Yongru Sun (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China). D. salina strain UTEX-1644 was purchased from the Algae Culture Collection at the University of Texas (Austin, USA) and cultured in the modified PKS medium under a light intensity of 50 mmol photon m s with a 12/12-h light/day [12]. To amplify the canstatin gene, the total RNA of human placental tissues was isolated. The Primer 1 (50-ACTCCC GGGGTCAGCATCGGCTACCTCCT-30) and Primer 2 (50-CCGAGCTCTCAATGGTGATGGTGATGGTG CA GGTTCTTCATGCACAC-30) were designed in which the Sma_ and Sac_ sites (underlined) were introduced respectively (the bold sequence represents the sequence of His6). Using the above RNA and primers, the human canstatin gene was amplified by reverse transcription–polymerase chain reaction (RT–PCR) and the results showed that a specific fragment of 700 bp was successfully amplified. The results of sequencing indicated that the amplified DNA fragment was completely identical to the nucleotide sequence reported in GenBank. Then, the canstatin gene was inserted into pMD18-T (TaKaRa, Dalian, China) to yield a new plasmid pMD18-T-Can for plasmid propagation and DNA sequencing. Subsequently, the canstatin fragment cut from the pMD18-T-Can by digestion of endonucleases was inserted into pUV-GUS to generate a novel vector pUV-Can. After being confirmed by digestion of double enzymes, the pUV-Can was recovered and further connected with the bar box to generate a eukaryotic expression vector pUV-Can-Bar. pUV-Can-Bar vectors were transformed into the D. salina cells using the glass beads method [12], and then the transformed D. salina cells were incubated for 24 h under dim light conditions. After the transformed D. salina cells were incubated for 2 weeks on a 1% agar plate containing 3 mg/l of phosphinothricin (PPT), the individual positive colonies were observed, among which four colonies were picked out and inoculated into the liquid medium with 3 mg/l of PPT for further selection. The resistant test of the transformants demonstrated that all the transformants survived from the following Acta Biochim Biophys Sin 2014, 46: 428–430 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu009. Advance Access Publication 23 March 2014