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Prospects and limitations of the rational engineering of fibrillar collagens
Author(s) -
Majsterek Ireneusz,
McAdams Erin,
Adachi Eijiro,
Dhume Shirish T.,
Fertala Andrzej
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0385103
Subject(s) - thermostability , chemistry , recombinant dna , biochemistry , protein engineering , procollagen peptidase , collagen helix , rational design , mutant , matrix metalloproteinase , triple helix , cleavage (geology) , tissue engineering , enzyme , microbiology and biotechnology , biology , peptide , stereochemistry , gene , genetics , paleontology , fracture (geology)
Recombinant collagens are attractive proteins for a number of biomedical applications. To date, significant progress was made in the large‐scale production of nonmodified recombinant collagens; however, engineering of novel collagen‐like proteins according to customized specifications has not been addressed. Herein we investigated the possibility of rational engineering of collagen‐like proteins with specifically assigned characteristics. We have genetically engineered two DNA constructs encoding multi‐D4 collagens defined as collagen‐like proteins, consisting primarily of a tandem of the collagen II D4 periods that correspond to the biologically active region. We have also attempted to decrease enzymatic degradation of novel collagen by mutating a matrix metalloproteinase 1 cleavage site present in the D4 period. We demonstrated that the recombinant collagen α‐chains consisting predominantly of the D4 period but lacking most of the other D periods found in native collagen fold into a typical collagen triple helix, and the novel procollagens are correctly processed by procollagen N‐proteinase and procollagen C‐proteinase. The nonmutated multi‐D4 collagen had a normal melting point of 41°C and a similar carbohydrate content as that of control. In contrast, the mutant multi‐D4 collagen had a markedly lower thermostability of 36°C and a significantly higher carbohydrate content. Both collagens were cleaved at multiple sites by matrix metalloproteinase 1, but the rate of hydrolysis of the mutant multi‐D4 collagen was lower. These results provide a basis for the rational engineering of collagenous proteins and identifying any undesirable consequences of altering the collagenous amino acid sequences.