A molecular approach to optimize hIFN α2b expression and secretion in Yarrowia lipolytica.
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.
Gasmi
Najla
N
INRA, UMR1319 Micalis, Domaine de Vilvert, 78352 Jouy-en-Josas, France.
Fudalej
Franck
F
Kallel
Héla
H
Nicaud
Jean-Marc
JM
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
D003062:Codon; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D021382:Protein Sorting Signals; D011994:Recombinant Proteins