Expression, purification and insights into structure and folding of the ADAM22 pro domain

• Published in: Protein expression and purification Vol. 61; no. 2; pp. 175 - 183
• Main Authors: Sørensen, Hans Peter, Jacobsen, Jonas, Nielbo, Steen, Poulsen, Flemming M., Wewer, Ulla M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2008
• Subjects: ADAM Proteins - biosynthesis; ADAM Proteins - chemistry; ADAM Proteins - isolation & purification; ADAM22; Circular Dichroism - methods; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Humans; Magnetic Resonance Spectroscopy - methods; Matrix Metalloproteinase 3 - chemistry; Metalloprotease; Nerve Tissue Proteins - biosynthesis; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - isolation & purification; NMR structure; Protein expression; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Reproducibility of Results; Temperature; ADAM22; Metalloprotease; NMR structure; Protein expression
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2008.05.020

The ADAMs (a disintegrin and metalloproteases) are an important class of enzymes in the regulation of human disease. The pro domains of ADAMs are responsible for the latency and secretion of mature enzymes. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme after secretion. To understand the functions of human ADAM pro domains and to determine three-dimensional structures, we have screened promising targets for expression and purification properties when using Escherichia coli as the host. The pro domain of ADAM22 (ADAM22-P) expressed in E. coli was folded, as determined by CD and NMR spectroscopy. An ADAM22-P fragment encoding residues 26–199 could be expressed in high amounts, remained soluble above 1 mM, and was suitable for structural studies by NMR spectroscopy. CD spectroscopy and predictions suggest that the secondary structure in ADAM22-P consists of β-strands. Furthermore, our data indicate that the pro domains of ADAMs are expressed as two subdomains. The most N-terminal subdomain (ADAM22-P N) was found to be susceptible to proteolysis and was required for folding stability of the second subdomain (ADAM22-P C).