PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE

KLEINE T, BARTSCH S, BLASER J, SCHNIERER S, TRIEBEL S, VALENTIN M, GOTE T, Tschesche H (1993)
BIOCHEMISTRY 32(51): 14125-14131.

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Abstract
TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The K(i) values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1. As for the PMNL-collagenase, the N-terminal catalytic domain is sufficient for enzyme-inhibitor interaction and binding.
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KLEINE T, BARTSCH S, BLASER J, et al. PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE. BIOCHEMISTRY. 1993;32(51):14125-14131.
KLEINE, T., BARTSCH, S., BLASER, J., SCHNIERER, S., TRIEBEL, S., VALENTIN, M., GOTE, T., et al. (1993). PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE. BIOCHEMISTRY, 32(51), 14125-14131.
KLEINE, T., BARTSCH, S., BLASER, J., SCHNIERER, S., TRIEBEL, S., VALENTIN, M., GOTE, T., and Tschesche, H. (1993). PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE. BIOCHEMISTRY 32, 14125-14131.
KLEINE, T., et al., 1993. PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE. BIOCHEMISTRY, 32(51), p 14125-14131.
T. KLEINE, et al., “PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE”, BIOCHEMISTRY, vol. 32, 1993, pp. 14125-14131.
KLEINE, T., BARTSCH, S., BLASER, J., SCHNIERER, S., TRIEBEL, S., VALENTIN, M., GOTE, T., Tschesche, H.: PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE. BIOCHEMISTRY. 32, 14125-14131 (1993).
KLEINE, T, BARTSCH, S, BLASER, J, SCHNIERER, S, TRIEBEL, S, VALENTIN, M, GOTE, T, and Tschesche, Harald. “PREPARATION OF ACTIVE RECOMBINANT TIMP-1 FROM ESCHERICHIA-COLI INCLUSION-BODIES AND COMPLEX-FORMATION WITH THE RECOMBINANT CATALYTIC DOMAIN OF PMNL-COLLAGENASE”. BIOCHEMISTRY 32.51 (1993): 14125-14131.
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