title: Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris.
abstract: 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.
authors: Centre for Biotechnology, Anna University, Chennai 600 025, India.
authors: Ashok Kumar Prasanna
authors: AK
authors: Vadhana
authors: Premsingh
authors: P
authors: Samuel
authors: Ronald M
authors: RM
authors: Berin
authors: Jayachandran
authors: J
authors: Krishna
authors: Kavitha
authors: K
authors: Kamatchi
authors: Sankaranarayanan
authors: S
authors: Meenakshisundaram
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019149:Bioreactors; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis / Q000235:genetics; D006801:Humans; D007218:Industrial Microbiology; D008049:Lipase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D061785:Transcription Initiation, Genetic
chemical_list: D004792:Enzyme Precursors; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D008049:Lipase; C431349:lipase B, Candida antarctica