Purification and characterization of a recombinant human cripto-1 protein

Growth Factors. 1998;15(3):215-29. doi: 10.3109/08977199809002118.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers, Tumor / chemistry
  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / isolation & purification*
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Epidermal Growth Factor*
  • Escherichia coli
  • Female
  • GPI-Linked Proteins
  • Growth Substances / chemistry
  • Growth Substances / genetics
  • Growth Substances / isolation & purification*
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Mammary Glands, Animal / chemistry
  • Membrane Glycoproteins*
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Neoplasm Proteins / chemistry
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / isolation & purification*
  • Protein Folding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Solubility
  • Sulfhydryl Reagents / metabolism

Substances

  • Biomarkers, Tumor
  • GPI-Linked Proteins
  • Growth Substances
  • Intercellular Signaling Peptides and Proteins
  • Membrane Glycoproteins
  • Neoplasm Proteins
  • Recombinant Proteins
  • Sulfhydryl Reagents
  • TDGF1 protein, human
  • Tdgf1 protein, mouse
  • Epidermal Growth Factor

Associated data

  • GENBANK/P13385
  • GENBANK/P51865