Polynucleotide and polypeptide sequence and methods thereof
AssigneeBigtec Private Limited
InventorsMulakkapurath Narayanan Manoj, Venkata Ramachandra Rao Vasamsetty, Madhuri Baliga, Kirubakaran Naveen Kumar, Chandrasekhar Bhaskaran Nair, Pillarisetti Venkata Subbarao
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably Pichia pastoris. The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins.
Claims18
An isolated nucleic acid comprising the polynucleotide sequence as set forth in SEQ ID NO:
- 2.
A method for obtaining a recombinant insulin precursor molecule having the polypeptide sequence as set forth in SEQ ID NO:
- 1, said method comprising steps of:
- a) synthesizing the polynucleotide sequence set forth in SEQ ID NO:
- 2 by combining 26 oligonucleotides of SEQ ID NOS:
- 3 to 28 by assembly PCR, and inserting the synthesized polynucleotide sequence in a vector,
- b) transforming a host cell with said vector followed by antibiotic screening host selection, and
- c) fermenting the selected transformed host cell and in-situ capturing of the insulin precursor molecule to obtain said precursor having the polypeptide sequence as set forth in SEQ ID NO:
- 1.
A method for obtaining a recombinant insulin molecule from a precursor molecule having the polypeptide sequence as set forth in SEQ ID NO:
- 1, said method comprising steps of:
- a) synthesizing the polynucleotide sequence set forth in SEQ ID NO:
- 2 by combining 26 oligonucleotides of SEQ ID NOS:
- 3 to 28 by assembly PCR,
- b) inserting the synthesized polynucleotide sequence in a vector and transforming a host cell with said vector followed by antibiotic screening host selection,
- c) fermenting the selected transformed host cell followed by downstream processing for in-situ capturing of the insulin precursor molecule, and
- d) in-situ conversion of the insulin precursor molecule having the polypeptide sequence as set forth in SEQ ID NO:
- 1 into said recombinant insulin molecule.
A recombinant vector comprising the polynucleotide sequence set forth in SEQ ID NO:
- 2.
The recombinant vector as claimed in 15, wherein the recombinant vector is selected from a group comprising pPIC9K and pPICZα, preferably pPIC9K.
A recombinant host cell, transformed by introduction of a vector comprising the polynucleotide sequence set forth in SEQ ID NO:
- 2.
Dependencies are inferred from the claim text.