title: Optimization of the recombinant production and purification of a self-assembling peptide in Escherichia coli.
abstract: BACKGROUND
  Amphiphilic peptides are important building blocks to generate nanostructured biomaterials for drug delivery and tissue engineering applications. We have shown that the self-assembling peptide SA2 (Ac-AAVVLLLWEE) can be recombinantly produced in E. coli when fused to the small ubiquitin-like modifier (SUMO) protein. Although this system yielded peptides of high purity with no residual amino acids after cleavage of the SUMO fusion protein, the yield after purification was generally low (~1 mg/L bacterial culture) as compared to other peptides and proteins produced with the same method and under the same conditions.
  
  
  RESULTS
  The aim of this study is to understand the underlying mechanisms causing the low yield of this recombinant peptide in E. coli and to optimize both production and purification of recombinant SA2 peptides. It was demonstrated that by simply changing the medium to a well-balanced auto-induction medium the yield of recombinant production was augmented (~4 fold). Moreover, it was demonstrated that self-assembly of SUMO-SA2 fusion proteins caused the low peptide yields after purification. By replacing the second IMAC purification step with a selective precipitation step, peptide yields could be increased approx. 3 fold. With these optimizations in place the overall yield of purified SA2 peptide increased with 12-fold.
  
  
  CONCLUSIONS
  Premature self-assembly of the SUMO-SA2 fusion construct interfered with proper purification of the SA2 peptide, resulting in low yields of purified peptide and this could be prevented by changing the mode of purification. These findings are important when setting up purification schemes for other self-assembling peptides with the use of a SUMO fusion construct.
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG, Utrecht, The Netherlands. m.radmalekshahi@uu.nl.|Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. m.radmalekshahi@uu.nl.
authors.forename: Mazda
authors.initials: M
authors.lastname: Rad-Malekshahi
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG, Utrecht, The Netherlands. Matthiasflement@hotmail.com.
authors.forename: Matthias
authors.initials: M
authors.lastname: Flement
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG, Utrecht, The Netherlands. W.E.Hennink@uu.nl.
authors.forename: Wim E
authors.initials: WE
authors.lastname: Hennink
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG, Utrecht, The Netherlands. E.Mastrobattista@uu.nl.
authors.forename: Enrico
authors.initials: E
authors.lastname: Mastrobattista
mesh_terms: D004926:Escherichia coli; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins