Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage.
Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.
Brockmann
Eeva-Christine
EC
Department of Biotechnology, University of Turku, Tykistökatu 6A 6, FIN-20520 Turku, Finland. eechbr@utu.fi
Lamminmäki
Urpo
U
Saviranta
Petri
P
D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics*; D003545:Cysteine / Q000235:genetics; D004094:Dihydropteroate Synthase / Q000032:analysis / Q000235:genetics*; D007118:Immunoassay; D009154:Mutation; D019151:Peptide Library; D019897:Periplasm / Q000201:enzymology; D033903:Periplasmic Proteins / Q000032:analysis; D015202:Protein Engineering
D019151:Peptide Library; D033903:Periplasmic Proteins; D001224:Aspartic Acid; D001216:Asparagine; D004094:Dihydropteroate Synthase; D003545:Cysteine