Purification and characterization of a recombinant human cripto-1 protein

Masaharu Seno, Marta Desantis, Subha Kannan, Caterina Bianco, Hiroko Tada, Nancy Kim, Megumi Kosaka, William J. Gullick, Hidenori Yamada, David S. Salomon

Research output: Contribution to journalArticle

29 Citations (Scopus)

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 β-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.

Original languageEnglish
Pages (from-to)215-229
Number of pages15
JournalGrowth Factors
Volume15
Issue number3
Publication statusPublished - 1998

Fingerprint

Purification
Proteins
human TDGF1 protein
Epidermal Growth Factor
Cysteine
Escherichia coli
Aptitude
Phosphorylation
Escherichia coli Proteins
Inclusion Bodies
Receptor Protein-Tyrosine Kinases
Reverse-Phase Chromatography
Protein Sorting Signals
Collagen Type I
Caseins
Recombinant Proteins
Histidine
Chromatography
Oxidation-Reduction
Tyrosine

Keywords

  • β-casein expression
  • 3-trimethylammoniopropyl methanethiosulfonate
  • Cripto
  • Duct-like branching
  • MAPK activation
  • Recombinant expression
  • Refolding
  • Shc phosphorylation

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Endocrinology
  • Cell Biology

Cite this

Seno, M., Desantis, M., Kannan, S., Bianco, C., Tada, H., Kim, N., ... Salomon, D. S. (1998). Purification and characterization of a recombinant human cripto-1 protein. Growth Factors, 15(3), 215-229.

Purification and characterization of a recombinant human cripto-1 protein. / Seno, Masaharu; Desantis, Marta; Kannan, Subha; Bianco, Caterina; Tada, Hiroko; Kim, Nancy; Kosaka, Megumi; Gullick, William J.; Yamada, Hidenori; Salomon, David S.

In: Growth Factors, Vol. 15, No. 3, 1998, p. 215-229.

Research output: Contribution to journalArticle

Seno, M, Desantis, M, Kannan, S, Bianco, C, Tada, H, Kim, N, Kosaka, M, Gullick, WJ, Yamada, H & Salomon, DS 1998, 'Purification and characterization of a recombinant human cripto-1 protein', Growth Factors, vol. 15, no. 3, pp. 215-229.
Seno M, Desantis M, Kannan S, Bianco C, Tada H, Kim N et al. Purification and characterization of a recombinant human cripto-1 protein. Growth Factors. 1998;15(3):215-229.
Seno, Masaharu ; Desantis, Marta ; Kannan, Subha ; Bianco, Caterina ; Tada, Hiroko ; Kim, Nancy ; Kosaka, Megumi ; Gullick, William J. ; Yamada, Hidenori ; Salomon, David S. / Purification and characterization of a recombinant human cripto-1 protein. In: Growth Factors. 1998 ; Vol. 15, No. 3. pp. 215-229.
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AU - Kosaka, Megumi

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