Synthetic gene for expressing Sm-14 in
The production of recombinant proteins using a synthetic gene for increased expression of the protein in Pichia pastoris is disclosed. More specifically, disclosed is a process for producing a recombinant Sm14 protein of Schistosoma mansoni , by providing a synthetic gene for increased production of Sm14 protein, producing the gene and genetically manipulating the Pichia pastoris strain with the gene in order to produce a vaccine effectively. Improved methods are also provided for producing and purifying this protein from P. pastoris cells, which can be scaled for industrial production.
1. A synthetic gene comprising SEQ ID NO:3.
2. A production process of recombinant Sm14 protein in Pichia pastoris , said process comprising:
(a) synthesis of a gene defined by SEQ ID NO:3;
(b) cloning of the synthesized gene in vector pPIC9K by BamHI site and reconstituting a Kozak sequence of gene AOX1 before a start codon of protein Sm14, for expression of Sm14 in its intracellular form; and
(c) transformation of P. pastoris with plasmid pPIC9K-Sm14-MV and selection of recombinant clones with multiple copies.
3. The process of claim 2 , further comprising:
(d) performing lysis of P. pastoris cells to obtain a lysate;
(e) clarifying the lysate obtained in stage (d) to obtain a clarified lysate;
(f) loading the clarified lysate in a column containing an anion-exchange resin and eluting proteins by pH changes in the column; and
(g) separating contaminant proteins from the recombinant Sm14 protein by gel-filtration.