Nucleotide sequence coding for a modified protein of interest, expression vector and method for obtaining same

A method for obtaining a purified and immobilized modified protein of interest, said protein of interest having, after purification and immobilization, at least the same biological activity as the native protein of interest and being directly usable, said method being characterized in that it comprises the following steps:

• at least two nucleotide sequences encoding said modified protein of interest, comprising at least one gene encoding said protein of interest, a “polyK” nucleotide fragment encoding a series of at least six lysine residues and a “polyH” nucleotide fragment encoding a series of at least six histidine residues, are provided, the two sequences, chosen from different groups, being chosen from:
• (a) the nucleotide sequences in which, with respect to the gene, the two nucleotide fragments, polyK or polyH, are located on the 5′ end of the sequence;
• (b) the sequences in which, with respect to the gene, one of the two nucleotide fragments, polyK or polyH, is located on the 5′ end of the sequence, and the other is located on the 3′ end;
• (c) the sequences in which, with respect to the gene, the two nucleotide fragments, polyK and polyH, are located on the 3′ end of the sequence;
• the nucleotide sequences are expressed in a suitable expression system;
• the modified proteins thus obtained are purified by metal ion affinity chromatography;
• the purified modified proteins are immobilized on a linear or particulate polymer;
• the biological activity of the immobilized modified proteins is tested;
• and
• the immobilized modified protein exhibiting the best biological activity is selected.