title: Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.
abstract: Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme fromYersinia mollaretii(Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.|School of Engineering and Science, Jacobs University Bremen, Bremen, Germany.
authors.forename: Amol V
authors.initials: AV
authors.lastname: Shivange
authors.affiliation: BASF SE, Fine Chemicals and Biocatalysis Research, Ludwigshafen, Germany.
authors.forename: Hans Wolfgang
authors.initials: HW
authors.lastname: Hoeffken
authors.affiliation: BASF SE, Fine Chemicals and Biocatalysis Research, Ludwigshafen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Haefner
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.|School of Engineering and Science, Jacobs University Bremen, Bremen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D016415:Sequence Alignment; D015007:Yersinia / Q000201:enzymology / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D010832:6-Phytase
keywords: ProCoS; directed evolution; mutagenesis; pH stability; phytase; protein engineering method; protein surface engineering