Team:Slovenia/Polypeptide manufacturing Idea Approach.html

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Polypeptide manufacturing

Summary
One of the main problems in manufacturing polypeptides is that the isolation and sometimes production method varies from protein to protein, depending on their properties. We prepared the required BioBricks and introduced a procedure that streamlines and unifies manufacturing of polypeptides which results, in principle independently of their sequence, in high yield polypeptide production in bacteria. We demonstrated that peptide production of which in bacteria is difficult due to proteolytic degradation of small unstructured polypeptides, its toxicity to bacteria, such as in antimicrobial peptides and difficult isolation due to small size, can be successfully and in high yield produced as a fusion partner of insoluble ketosteroid isomerase (KSI). Further, acidic cleavage of an Asp-Pro dipeptide between KSI carrier and desired polypeptide is a simple and efficient way for releasing the preferred polypeptide. Since the KSI remains insoluble after cleavage, polypeptide can be easily isolated with HPLC.

The idea & Approach
Usually for every polypeptide production, isolation and purification procedures have to be developed and optimized. For peptides high-yield expression in host such as bacteria is very difficult in form of a free polypeptide due to proteolytic degradation and difficult isolation due to small size. Examples of problematic production are also polypeptides toxic to their host where a lot of optimization is required to obtain small amounts of product. Our aim was to simplify and unify polypeptide manufacturing within the BioBrick system, enabling high-yield production of polypeptides regardless of their primary structure and properties. We addressed this problem using ketosteroid isomerase (KSI) as a fusion partner of our constructs (Kuliopulos et al, 1994). Strong promoter such as T7 drives expression of KSI together with fused polypeptide biobrick to insoluble inclusion bodies. Inclusion bodies are stable against proteolysis and can be easily purified as the insoluble bacterial lysate may already consist of more than 80 % of fusion protein. We also introduced an acid-labile Asp-Pro bond between KSI and polypeptide biobrick to enable separation of polypeptide from KSI (Figure 1), without having to use aggressive and toxic chemicals, such as CNBr that may modify labile amino acid residues (Zorko et al, 2009). After acidic cleavage the released polypeptide is additionally purified by HPLC. This procedure enables simple high yield production and purification of versatile polypeptides and most importantly it occurs under the same denaturing conditions for every polypeptide.  Figure 1: Principle of a simple and uniform production of polypeptide biobricks. Polypeptide biobricks are produced as a fusion partner with insoluble ketosteroid isomerase (KSI) in form of inclusion bodies. The linker between KSI and polypeptide biobrick contains acid-labile bond Asp-Pro enabling easy cleavage by acid hydrolysis and separation of polypeptide biobrick from KSI. His-tag may aid in additional initial purification of inclusion bodies or removal of KSI segment in case that the polypeptide is also insoluble under the native conditions and detection of bacterial product by Western blot.  Results 

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