1887

Abstract

The β-hexosyltransferase (BHT) from is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1–110), which included two predicted endogenous structural domains. The first domain (amino acids 1–22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23–110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant strains carrying combinations of the predicted structural domains and the α mating factor (MFα) from as positive control. Upon replacement of the leader domain (amino acids 1–22) by MFα (α- ), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53- and 14-fold, respectively. Leader interference was demonstrated when MFα preceded the putative classical rBHT leader (amino acids 1–22), explaining the limited secretion of soluble protein by (GS115 : : α- ). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-β-galactosidase single-chain variable antibody fragment scFv13R4. The recombinants carrying chimeras of the N-terminal 1–110 regions of rBHT preceding correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : α- -HIS and GS115 : : α- ) showed comparable activity rates of GOS generation.

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2016-01-01
2022-01-18
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References

  1. Bach H., Mazor Y., Shaky S., Shoham-Lev A., Berdichevsky Y., Gutnick D. L., Benhar I. 2001; Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. J Mol Biol 312:79–93 [View Article][PubMed]
    [Google Scholar]
  2. Bendtsen J. D., Jensen L. J., Blom N., Von Heijne G., Brunak S. 2004; Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Sel 17:349–356 [View Article][PubMed]
    [Google Scholar]
  3. Blakely J. A., MacKenzie S. L. 1969; Purification and properties of a β-hexosidase from Sporobolomyces singularis . Can J Biochem 47:1021–1025 [View Article][PubMed]
    [Google Scholar]
  4. Boyd D., Beckwith J. 1990; The role of charged amino acids in the localization of secreted and membrane proteins. Cell 62:1031–1033 [View Article][PubMed]
    [Google Scholar]
  5. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
    [Google Scholar]
  6. Cereghino J. L., Cregg J. M. 2000; Heterologous protein expression in the methylotrophic yeast Pichia pastoris . FEMS Microbiol Rev 24:45–66 [View Article][PubMed]
    [Google Scholar]
  7. Cho Y. J., Shin H. J., Bucke C. 2003; Purification and biochemical properties of a galactooligosaccharide producing β-galactosidase from Bullera singularis . Biotechnol Lett 25:2107–2111 [View Article][PubMed]
    [Google Scholar]
  8. Coeytaux K., Poupon A. 2005; Prediction of unfolded segments in a protein sequence based on amino acid composition. Bioinformatics 21:1891–1900 [View Article][PubMed]
    [Google Scholar]
  9. Dagher S. F., Azcarate-Peril M. A., Bruno-Bárcena J. M. 2013; Heterologous expression of a bioactive β-hexosyltransferase, an enzyme producer of prebiotics, from Sporobolomyces singularis . Appl Environ Microbiol 79:1241–1249 [View Article][PubMed]
    [Google Scholar]
  10. Damasceno L. M., Huang C. J., Batt C. A. 2012; Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93:31–39 [View Article][PubMed]
    [Google Scholar]
  11. Gorin P.A.J., Phaff H. J., Spencer J.F.T. 1964a; The structures of galactosyl-lactose and galactobiosyl-lactose produced from lactose by Sporobolomyces singularis . Can J Chem 42:1341–1344 [View Article]
    [Google Scholar]
  12. Gorin P.A.J., Spencer J.F.T., Phaff H. J. 1964b; The synthesis of β-galacto-β-gluco-pyranosyl disaccharides by Sporobolomyces singularis . Can J Chem 42:2307–2317 [View Article]
    [Google Scholar]
  13. Gosling A., Stevens G. W., Barber A. R., Kentish S. E., Gras S. L. 2010; Recent advances refining galactooligosaccharide production from lactose. Food Chem 121:307–318 [View Article]
    [Google Scholar]
  14. Grage K., Rehm B.H.A. 2008; In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner. Bioconjug Chem 19:254–262 [View Article][PubMed]
    [Google Scholar]
  15. Gupta R., Brunak S. 2002; Prediction of glycosylation across the human proteome and the correlation to protein function. In Pacific Symposium on Biocomputing vol 7 pp 310–322 Edited by Altman R. B., Dunker A. K., Hunter L., Lauderdale K., Klein T. E. Singapore: World Scientific Publishing;
    [Google Scholar]
  16. Illanes A. 2011; Whey upgrading by enzyme biocatalysis. Electron J Biotechnol 14:1–42 [View Article]
    [Google Scholar]
  17. Ishikawa E., Sakai T., Ikemura H., Matsumoto K., Abe H. 2005; Identification, cloning, and characterization of a Sporobolomyces singularis β-galactosidase-like enzyme involved in galacto-oligosaccharide production. J Biosci Bioeng 99:331–339 [View Article][PubMed]
    [Google Scholar]
  18. Letunic I., Doerks T., Bork P. 2012; SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res 40:(D1)D302–D305 [View Article][PubMed]
    [Google Scholar]
  19. Martineau P., Jones P., Winter G. 1998; Expression of an antibody fragment at high levels in the bacterial cytoplasm. J Mol Biol 280:117–127 [View Article][PubMed]
    [Google Scholar]
  20. Panesar P. S., Panesar R., Singh R. S., Kennedy J. F., Kumar H. 2006; Microbial production, immobilization and applications of β-d-galactosidase. J Chem Technol Biotechnol 81:530–543 [View Article]
    [Google Scholar]
  21. Petersen T. N., Brunak S., von Heijne G., Nielsen H. 2011; SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786 [View Article][PubMed]
    [Google Scholar]
  22. Phaff H. J., do Carmo-Sousa L. D. 1962; Four new species of yeast isolated from insect frass in bark of Tsuga heterophylla (Raf.) Sargent. Antonie van Leeuwenhoek 28:193–207 [View Article][PubMed]
    [Google Scholar]
  23. Punta M., Forrest L. R., Bigelow H., Kernytsky A., Liu J., Rost B. 2007; Membrane protein prediction methods. Methods 41:460–474 [View Article][PubMed]
    [Google Scholar]
  24. Sakai T., Tsuji H., Shibata S., Hayakawa K., Matsumoto K. 2008; Repeated-batch production of galactooligosaccharides from lactose at high concentration by using alginate-immobilized cells of Sporobolomyces singularis YIT 10047. J Gen Appl Microbiol 54:285–293 [View Article][PubMed]
    [Google Scholar]
  25. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  26. Sears P., Wong C. H. 2001; Toward automated synthesis of oligosaccharides and glycoproteins. Science 291:2344–2350 [View Article][PubMed]
    [Google Scholar]
  27. Shin H. J., Yang J. W. 1998; Enzymatic production of galactooligosaccharide by Bullera singularis β-galactosidase. J Microbiol Biotechnol 8:484–489
    [Google Scholar]
  28. Shin H. J., Park J. M., Yang J. W. 1998; Continuous production of galacto-oligosaccharides from lactose by Bullera singularis β-galactosidase immobilized in chitosan beads. Process Biochem 33:787–792 [View Article]
    [Google Scholar]
  29. Spencer J.F.T., Ragout de Spencer A. L., Laluce C. 2002; Non-conventional yeasts. Appl Microbiol Biotechnol 58:147–156 [View Article][PubMed]
    [Google Scholar]
  30. Tzortzis G., Vulevic J. 2009; Galacto-oligosaccharide prebiotics. In Prebiotics and Probiotics Science and Technology pp 207–244 Edited by Charalampopoulos D., Rastall R. New York: [View Article] Springer;
    [Google Scholar]
  31. Visintin M., Tse E., Axelson H., Rabbitts T. H., Cattaneo A. 1999; Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc Natl Acad Sci U S A 96:11723–11728 [View Article][PubMed]
    [Google Scholar]
  32. von Heijne G. 1983; Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133:17–21 [View Article][PubMed]
    [Google Scholar]
  33. Wootton J. C. 1994; Non-globular domains in protein sequences: automated segmentation using complexity measures. Comput Chem 18:269–285 [View Article][PubMed]
    [Google Scholar]
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