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High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris


Human α-defensin 5 (HD5), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD5 through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD5 mature peptide (rmHD5) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD5 by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD5 into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD5 vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD5 nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD5, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD5. The results showed that about 165.0 mg/l of rmHD5 with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD5 was recovered after purification. The in vitro experiments revealed that rmHD5 exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD5 in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.

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This work was supported by grants from the National Natural Science Foundation of China (no. 30771892), Academician Fund of Chongqing City (no. CSTC, 2007AB5022), Special Fund of National Key Laboratory of Trauma, Burn and Combined Injury (no. SKLZZ200821), Natural Science Foundation of Chongqing City (no. CSTC, 2008BB5137), and National “863” High-tech Development Plan of China (no. 2007AA02Z152).

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Correspondence to Yongping Su or Junping Wang.

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Wang, A., Wang, S., Shen, M. et al. High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris . Appl Microbiol Biotechnol 84, 877–884 (2009).

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  • Human α defensin 5
  • Pichia pastoris
  • Expression
  • Purification
  • Bioactivity