Purification and characterization of a recombinant human cripto-1 protein.
Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.
Seno
M
M
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Japan. senom@biotech.okayamau.ac.jp
DeSantis
M
M
Kannan
S
S
Bianco
C
C
Tada
H
H
Kim
N
N
Kosaka
M
M
Gullick
W J
WJ
Yamada
H
H
Salomon
D S
DS
D000818:Animals; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor; D004926:Escherichia coli; D005260:Female; D058851:GPI-Linked Proteins; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008321:Mammary Glands, Animal / Q000737:chemistry; D008562:Membrane Glycoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013439:Sulfhydryl Reagents / Q000378:metabolism
D014408:Biomarkers, Tumor; D058851:GPI-Linked Proteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C070636:TDGF1 protein, human; C489450:Tdgf1 protein, mouse; D004815:Epidermal Growth Factor