Production and purification of self-assembling peptides in Ralstonia eutropha.
Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.
Reed
David C
DC
Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755, USA.
Barnard
Gavin C
GC
Anderson
Errik B
EB
Klein
Linden T
LT
Gerngross
Tillman U
TU
D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D020118:Cupriavidus necator / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D023822:Tissue Engineering / Q000379:methods
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins