Recombinant Dermatophagoides farinae type 1 allergen protein and its preparation method and application
Inventors
MA, Bruce Yong • Fan, Yu • Wang, Jun • WANG, Anliang
Assignees
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Publication Number
US-10975128-B2
Publication Date
2021-04-13
Expiration Date
Abstract
Provided are an optimized proDer f1 gene, a proDer f1 protein encoded thereby, a vector comprising said gene, and a Pichia pastoris strain. Also provided are an expression method and a purification method of the proDer f1 protein.
Core Innovation
The invention relates to producing recombinant Dermatophagoides farinae type 1 allergen (Der f1) in the Pichia pastoris system. It provides a codon-optimized proDer f1 gene having a base sequence shown in SEQ ID NO:1, encoding proDer f1 and used in vectors for expression in Pichia pastoris.
The disclosed design emphasizes optimizing gene expression by improving CAI from 0.76 to 0.85, eliminating low-utility codons, refining GC distribution, and integrating expression elements near the AOX1 ATG with an approximately 242 bp spacing. It further includes an alpha-factor signal peptide plus a Kozak sequence GCCACCATGG preceding the proDer f1 coding region.
The invention reports that purified recombinant Der f1 is functionally similar to natural Der f1 based on patient serum reactivity. It also provides an expression and purification approach that yields purified mature Der f1 protein with a reported yield of at least 200 mg/L.
Claims Coverage
The independent claim is directed to an isolated DNA sequence encoding the proDer f1 protein having the base sequence shown in SEQ ID NO:1, and the DNA sequence is comprised in a vector. Across the identified claims, the coverage includes at least three inventive arrangements: sequence-in-vector, vector selection, and host/arrangement features including positional and regulatory elements.
Isolated DNA encoding proDer f1 in a vector
An isolated DNA sequence encoding a propeptide of Der f1 protein (proDer f1 protein) having a base sequence as shown in SEQ ID NO:1, wherein the DNA sequence is comprised in a vector.
Selected expression vectors containing the proDer f1 DNA
A DNA sequence of the invention wherein the DNA sequence is comprised in a vector selected from pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ A, B, C, or pGAPZ A, B, C.
Specific Pichia pastoris strain context
A DNA sequence of the invention wherein the DNA sequence is comprised in a vector that is present within Pichia pastoris strains selected from SMD1168, GS115, KM71, X33, and KM71H.
AOX1-proximal arrangement with alpha-factor signal peptide and Kozak
A DNA sequence of the invention in Pichia pastoris wherein the DNA arrangement includes an approximately 242 bp interval between the proDer f1-encoding region and the ATG of AOX1, and wherein the proDer f1-encoding DNA is preceded by an alpha-factor signal peptide and a Kozak sequence GCCACCATGG.
Overall, the claim coverage centers on an isolated SEQ ID NO:1 DNA encoding proDer f1 integrated into a vector, with further limitations relating to named vectors and Pichia pastoris strains, and with a defined regulatory and positional arrangement near AOX1 including an approximately 242 bp interval, an alpha-factor signal peptide, and the Kozak sequence GCCACCATGG.
Stated Advantages
Purified recombinant Der f1 is functionally similar to natural Der f1 based on patient serum reactivity.
Reported purification/expression approach provides a yield of at least 200 mg/L purified mature Der f1 protein.
Documented Applications
Medicament use for dust mite allergic disease (allergic rhinitis, allergic asthma).
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