Polypeptide manufacturing Results.html

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Polypeptide manufacturing

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 4) 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. }}