## 3 Results and Discussion  
### Production and Purification of Recombinant AMP-A200 and AMP-SELP  
All recombinant proteins (Hep-A200, Syn-A200, Hep-SELP, Syn-SELP, CM4-SELP and BMAP18-SELP) were successfully produced in _E. coli_ BL21(DE3) at high expression levels (see Figures 1b and 2a for representative samples).  
Purification of the A200-based protein polymers was achieved by ITC, resulting in highly pure protein fractions (Figure 1). After three heating/cooling cycles (Figure 1a), a pure protein fraction was obtained, as demonstrated by presence of a single band around 100 kDa (lane 7, Figure 1b).  
Figure 1: Purification of AMP-A200-based protein polymers. (**a**) Schematic representation of the purification process by ITC. (**b**) Representative SDS-PAGE analysis of purification for Syn-A200. After sonication and acidification to pH 3.5, the insoluble debris was removed by centrifugation and the recombinant protein polymer was purified from the clear supernatant (lane 1) by ITC using hot/cold cycles. For each cycle, the protein-enriched solution was heated to 37 \({}^{\circ}\)C and then centrifuged at the same temperature. The resulting hot supernatant was discarded (lanes 2, 4 and 6) and the precipitated protein was resuspended in ice-cold ddH2O. After complete resuspension, the solution was centrifuged at 4 \({}^{\circ}\)C, and the resulting cold supernatant (lanes 3, 5 and 7) was submitted to a new cycle. A pure protein polymer fraction (indicated by the white arrow) was obtained after three complete cycles. MWM—molecular weight marker (Precision Plus Protein Unstained Standards from Bio-Rad) in kDa; 1—cycle one, cold supernatant; 2—cycle one, hot supernatant; 3—cycle two, cold supernatant; 4—cycle two, hot supernatant; 5—cycle three, cold supernatant; 6—cycle three, hot supernatant. Gel was stained with 0.3 M CuCl2.  
Figure 2: (**a**) Representative SDS-PAGE analysis of the purification process for BMAP18-SELP by salting out with ammonium sulfate at 20% saturation: MWM—molecular weight marker (GRS Unstained Protein Marker from Grisp) in kDa; 1—crude cell lysate after sonication; 2—supernatant after acidification at pH 3.5; 3—supernatant after salting out; 4—fraction of the precipitated sample after salting out; 5—dialyzed protein sample. Gel was stained with 0.3 M CuCl2. (**b**) Representative SDS-PAGE (image on the left) and Western blot (image on the right) analysis of pure BMAP18-SELP (6) with unstained SDS-PAGE standard molecular weight marker (MWM, in kDa; BioRad).  
Purification of AMP-SELP was achieved by a non-chromatographic method involving the use of ammonium sulfate at 20% saturation for the selective precipitation of SELP [6]. Analysis of the electrophoretic pattern of purified BMAP18-SELP (used in Figure 2 as representative sample) revealed the presence of two strong bands at approximately 60 and 80 kDa and some less intense bands at lower molecular weights (Figure 2a). Western blot analysis using antibodies to the His tag revealed a similar band profile (Figure 2b), characterized by a strong band corresponding to BMAP18-SELP and unknown lower molecular weight protein bands. Overall, ammonium sulfate precipitation demonstrably resulted in pure AMP-SELP fractions.  
The same electrophoretic pattern and Western blot results were observed for the remaining SELP-based constructions (Figure S1). The abnormal gel mobility to higher molecular weights than expected is a consequence of the hydrophobic nature of the proteins and has been previously observed for other recombinant protein polymers [6; 45; 46; 59; 60]. After purification and lyophilization, the lyophilized product was weighed, resulting in volumetric productivities per liter of production of 50 mg/L for Hep-A200, 90 mg/L for Syn-A200, 90 mg/L for Hep-SELP, 73 mg/L for Syn-SELP, 120 mg/L for CM4-SELP and 70 mg/L for BMAP18-SELP.