Polypeptide manufacturing.html

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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

Several polypeptide constructs that were not likely to be efficiently produced in the free form were prepared as KSI fusions to show the feasibility of our approach (Figure 1).

Figure 1: Some of the constructs that were small unstructured peptides or that contained antimicrobial segments were prepared as fusions with KSI.
To demonstrate the described principle we prepared a BioBrick expressing small antimicrobial peptide fused to the C-terminal end of KSI via Asp-Pro (DP) linker. The coding sequence was cloned into the vector that adds His-tag at the N-terminus of the sequence according to our biobrick standard. KSI-peptide fusion polypeptide was then expressed in bacteria E.coli BL21 (DE3) pLysS and purified.

The procedure of isolating peptide from the inclusion bodies is carried out as illustrated in the flow chart (Figure 2).

Figure 2: Flow chart of the acidic cleavage of the fusion polypeptide and its purification.
Recombinant fusion protein was expressed in the form of inclusion bodies (IB) which were washed with lysis buffer, 2 M urea and MQ water and dissolved in the denaturant Gdn HCl to remove non-protein contaminants. Dissolved inclusion bodies were dialyzed against MQ. SDS-PAGE shows that fusion protein was present in IB and not in the soluble fraction (Figure 3).

Figure 3: Isolation of KSI-DP-P2/33 SDS-PAGE of cell lysate supernatant (lane 2) and IB (lane 3) shows that KSI-DP-P2/33 (Mw of approximately 14.6 kDa) is present mainly in inclusion bodies.
During dialysis the fusion protein precipitated. The precipitate was resuspended in 90 mM HCl and incubated at 85° C for 2 h. During this step a selective hydrolysis of Asp-Pro bond between KSI and antimicrobial peptide occurred and our peptide was separated from the KSI. After cleavage the antimicrobial peptide became soluble while KSI remained insoluble. Another dialysis against MQ was necessary to eliminate the salt that was obtained as a product of neutralization of HCl with NaOH. The antimicrobial peptide was further purified by HPLC (Figure 5) and its identity was confirmed by mass spectrometry.

Figure 4: Chromatograph of the sample after cleavage. HPLC was performed under the following conditions: buffer A: 5% acetonitrile, 5mM HCl, buffer B: 95%, acetonitrile, 5 mM HCl, using Jupiter C12 column.
We have demonstrated that peptide whose production in bacteria is difficult due to rapid degradation and its antimicrobial activity can be successfully and in high yield produced as a fusion partner of insoluble KSI. Further, acidic cleavage is a simple and efficient way for separating fused polypeptides and since KSI remains insoluble after cleavage, short peptide can be easily isolated with HPLC.

Our procedure represents an alternative production of polypeptides since it attempts to generalize manufacturing of polypeptides that allows, in principle independently of their sequence, high yield production in bacteria regardless of the toxicity, sensitivity to proteolysis and minimizes the purification steps.