Bone morphogenetic protein-1: The yype I procollagen C-proteinase

Science

Volumn 271, Issue 5247, 1996, Pages 360-362

  Kessler, Efrat ^a   Takahara, Kazuhiko ^b   Biniaminov, Luba ^a   Brusel, Marina ^a   Greenspan, Daniel S ^b  

^a TEL AVIV UNIVERSITY   (Israel)

^b UNIVERSITY OF WISCONSIN SCHOOL OF MEDICINE AND PUBLIC HEALTH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BONE MORPHOGENETIC PROTEIN; METALLOPROTEINASE; PEPTIDE FRAGMENT; PROCOLLAGEN; PROCOLLAGEN C PROTEINASE; PROCOLLAGEN TYPE I CARBOXY TERMINAL PEPTIDE; PROCOLLAGEN TYPE II CARBOXY TERMINAL PEPTIDE; PROCOLLAGEN TYPE II CARBOXY-TERMINAL PEPTIDE; PROTEIN; RECOMBINANT PROTEIN;

AMINO ACID SEQUENCE; ANIMAL; ARTICLE; CHEMISTRY; HUMAN; METABOLISM; MOLECULAR GENETICS; MOLECULAR WEIGHT; MOUSE;

AMINO ACID SEQUENCE; ANIMALS; BONE MORPHOGENETIC PROTEINS; HUMANS; METALLOENDOPEPTIDASES; MICE; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; PEPTIDE FRAGMENTS; PROCOLLAGEN; PROTEINS; RECOMBINANT PROTEINS;

ANIMALIA;

EID: 0030593032     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5247.360     Document Type: Article

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21. Procollagens, PCP, and PCPE were prepared as described (11, 12) [E. Kessler and B. Goldberg, Anal Biochem 86, 463 (1978)] Enzyme digests and SDS-PAGE analyses (in 6 and 12% gels for unreduced and reduced samples, respectively) were as described [S.-T Lee, E Kessler, D S. Greenspan, J. Biol. Chem. 265, 21992 (1990)].
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24. 2-terminal amino acid sequencing was performed with the ABI 475A liquid phase protein sequenator.
25. 2-terminal amino acid sequencing was performed with the ABI 475A liquid phase protein sequenator.
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27. For Fig 3A, proteins in the media of insect cells were concentrated by TCA precipitation (12). For Fig 3B, affinity-purified rBMP-1 and PCP were incubated (3 hours; 37°C) with PNGase F (Now England Biolabs, 140 ng) in buffer A containing SBTI (100 μg/ml), benzamidine (10 mM), leupeptin (10 μg/ml), SDS (0.1%), and NP-40 (1%). Immunoblots were developed with guinea pig antiserum to BMP-1 fusion protein (1 1000), with the ECL system (Amersham) To prepare BMP-1 fusion protein, we subcloned a 1040-bp Apa I-Hinc II cDNA fragment into expression vector pRSET B (Invitrogen) and expressed it in BL21 (DE3) Escherichia coli. The fusion protein, which includes human BMP-1 residues 197 to 543 (1) fused to a polyhistidine domain, was purified on nickel-Sepharose (Invitrogen) followed by SDS-PAGE (11) Gel suspensions in phosphate-buffered saline (11) containing 15 (first immunization), 7 5 (second and third boosts), or 20 μg of fusion protein (fourth boost) were injected subcutaneously into a guinea pig at 2-week intervals. Sera were collected 10 days after the third and fourth immunizations.
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30. 2-terminal sequencing as in Fig 1D.
31. 2-terminal sequencing as in Fig 1D.
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37. 2-terminal amino acid sequencing and S. Kotev-Emeth for assistance with figures preparation. This work was supported by grant 89-00498/1 (E K. and D S.G.) from the United States-Israel Binational Science Foundation, Jerusalem, Israel, and by NIH grant GM46846 (D.S.G.)