Display of Ras on filamentous phage through cysteine replacement.
Phage display technology has been used in a variety of contexts to understand and manipulate biomolecular interactions between proteins and other biomolecules. In this paper we describe the establishment of a phage display system for elucidation of the interactions between the GTPase Ras and its panel of effectors. It is shown how technical problems associated with phage display of a protein with unpaired cysteines, likely to be caused by the oxidizing environment of the bacterial periplasm into which the protein is directed, can be overcome by cysteine replacement based on functional and structural studies. First, the catalytic domain (residues 1-166) of mammalian H-Ras (Ras) was observed to be displayed on phage in an incorrect conformation not detectable by antibodies recognizing conformational epitopes on Ras. Although truncation of the phage coat protein used as fusion partner (g3p) resulted in minor improvements in the display, Ras was tailored for phage display by cysteine replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting fusion-phage is recognized by the conformation-dependent anti-Ras antibodies. Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data pave the way for analysis of Ras-effector interactions using phage display technology yet demonstrate that phage display of proteins with normally reduced cysteines should be approached with caution.
Wind
T
T
University of Aarhus, Department of Molecular and Structural Biology, Denmark.
Kjaer
S
S
Clark
B F
BF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003090:Coliphages / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D020798:Two-Hybrid System Techniques; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019908:Proto-Oncogene Proteins c-raf; C489014:RAS1 protein, S cerevisiae; D018631:ras Proteins; D003545:Cysteine