title: A molecular approach to optimize hIFN α2b expression and secretion in Yarrowia lipolytica.
abstract: In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): pre-proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre-proLIP2 signal secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular secretion of small heterologous proteins in Yarrowia lipolytica.
authors.affiliation: INRA, UMR1319 Micalis, Domaine de Vilvert, 78352 Jouy-en-Josas, France.
authors.forename: Najla
authors.initials: N
authors.lastname: Gasmi
authors.forename: Franck
authors.initials: F
authors.lastname: Fudalej
authors.forename: Héla
authors.initials: H
authors.lastname: Kallel
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D021382:Protein Sorting Signals; D021381:Protein Transport; D011994:Recombinant Proteins; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D021382:Protein Sorting Signals; D011994:Recombinant Proteins