Recombinant self-assembling peptide examples

Panitch et al. (1997) produced impressive yields of 700 mg/L by E. coli fermentation of a simple small artificial protein comprising 60 repeats of an (AG)4 motif with 23 and 33 amino acid N- and C-terminal fusions, respectively. The (AG)240 repeat units were not separated after expression, and the fusion sequences comprised just 10% of the whole fusion protein. This meant that loss of polypeptide material due to inefficient cleavage by 

CNBr was negligible and resulted in around 78% yield recovery. The fed batch fermentation allowed the control of oxygen, nitrogen, and glucose sources, improving yields from tens of milligrams per liter previously obtained in standard batch fermentation. 

Reed et al. (2006) reported on the production of a self-assembling peptide using R. eutropha. This peptide, RADI6, was 16 amino acids in length and produced as tandem repeats with the sequence 

The introduction of a glutamic acid (E bold), after each RADI 6 repeat was made to allow cleavage by endoproteinase Glu-C, which cleaves C-terminal to glutamate. A cellulose binding domain (CBD) (Table 1) was selected as the affinity tag, as CBDs bind strongly and specifically to cellulose which is a relatively cheap and abundant purification matrix. The main problem encountered was the low level of peptide recovered. Theoretically, 1 g of fusion protein should give 267mg of peptide, but only 10.1 mg of peptide was recovered after RP-HPLC. 

The recombinant system for peptide production involved designing synthetic oligonucleotides, which could be annealed, strand extended, and then cloned into the pET-SUMO vector to generate a fusion protein. This fusion protein was expressed in E. coli BL21(DE3) cells by IPTG induction and then cells lysed by freeze-thaw lysis followed by sonication. The lysate was passed 

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