An attempt to substitute the cell binding domain of human fibronectin in lambda phage J protein: computer design and expression.
We superimposed hydropathic indexes of the human fibronectin cell binding domain (CBD) on the lambda phage J protein by computer, and substituted 22 amino acids from the fibronectin CBD for a part of the lambda phage J protein. The fibronectin cell binding domain -Arg-Gly-Asp-Ser- (-RGDS-) localizes at the junction of hydrophobicity and hydrophilicity. We selected a similar hydrophobic-to-hydrophilic junction in the J protein region for substitution. This junction corresponds to 150 bp of the PstI fragment of J protein cDNA. We synthesized 150 bp of the relevant PstI fragment that includes the cell binding domain region. The region was then constructed by serial cloning as an expression vector, pJCBD. The vector pJCBD expressed the fused protein named JCBD (M(r) 32 kDa) in E coli XL1-BLUE. The expressed JCBD protein was identified by Western blot analysis in the extract of the pJCBD carrying bacterial lysate using both rabbit anti-lambda phage antiserum and anti-CBD of fibronectin antibody. The JCBD protein appeared to recognize retinoblast cell membrane RGDS-directed receptors, detected by enzyme-linked immuno-sorbent assay and also by binding competition assay with synthetic pentapeptides, Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES). The former competitor inhibited completely fibronectin CBD-dependent binding activity of JCBD, the latter had no inhibitory activity. These results suggest that certain functional proteins engineered by computer design between human fibronectin cell binding domain and lambda phage J protein can be produced.
Shibata
K
K
Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
Mita
T
T
Nakamura
H
H
Yamashiro
K
K
Gotoh
S
S
Hiranuma
K
K
Higashi
K
K
Hirano
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002477:Cells / Q000378:metabolism*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics
D005353:Fibronectins; C083033:J protein, Bacteriophage lambda; D011993:Recombinant Fusion Proteins; D017376:Viral Tail Proteins