Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris
Autor: | Ashok Kumar Prasanna Vadhana, Kavitha Kamatchi, Ronald M Berin, Sankaranarayanan Meenakshisundaram, Premsingh Samuel, Jayachandran Krishna |
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Rok vydání: | 2013 |
Předmět: |
Signal peptide
Recombinant Fusion Proteins Molecular Sequence Data Gene Dosage Heterologous Bioengineering Peptide Protein Sorting Signals Real-Time Polymerase Chain Reaction Applied Microbiology and Biotechnology Biochemistry Pichia Pichia pastoris law.invention Fungal Proteins Industrial Microbiology Bioreactors Genes Reporter law Humans Amino Acid Sequence RNA Messenger Cloning Molecular Lipase Transcription Initiation Genetic Candida chemistry.chemical_classification Enzyme Precursors Base Sequence Sequence Homology Amino Acid biology Granulocyte-Macrophage Colony-Stimulating Factor RNA Fungal biology.organism_classification Molecular biology Enzyme chemistry Recombinant DNA biology.protein Nucleic Acid Conformation Candida antarctica Sequence Alignment Biotechnology |
Zdroj: | Enzyme and Microbial Technology. 52:177-183 |
ISSN: | 0141-0229 |
Popis: | Secretion efficiency of the 85-amino acid Sacchromyces cerevisiae alpha signal peptide and the 25-amino acid Candida antarctica lipase B signal (nsB) peptide were compared. Three reporter proteins used for the study are C. antarctica lipase A (CalA), lipase B (CalB) and hGMCSF. The copy number of recombinant α-CalB and nsB-CalB clones was determined by qPCR and clones with equivalent gene copies were used for comparative analysis. About threefold increased CalB production corresponding to an activity of 480 U ml−1 was obtained with its native signal peptide, whereas with the alpha signal peptide the maximum activity was 160 U ml−1. Also, CalB was secreted as a mature protein with native N-terminus when fused to its own signal peptide, while unprocessed CalB with N-terminal extension was detected with the alpha signal peptide. Real time PCR analysis of CalB strains indicated that the difference in protein expression was not at the transcriptional level. The nsB signal sequence was also effective in secreting CalA enzyme and its secretion efficiency was on par with the alpha signal sequence. Further, hGMCSF fused inframe with the nsB signal peptide was also efficiently secreted into the medium. These results indicate that the nsB signal peptide can be a better alternative to alpha signal peptide for heterologous protein expression in Pichia pastoris. |
Databáze: | OpenAIRE |
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