Constructs for the expression of repeating triple-helical protein domains.
The development of novel scaffolds will be an important aspect in future success of tissue engineering. Scaffolds will preferably contain information that directs the cellular content of constructs so that the new tissue that is formed is closely aligned in structure, composition and function to the target natural tissue. One way of approaching this will be the development of novel protein-based constructs that contain one or more repeats of functional elements derived from various proteins. In the present case, we describe a strategy to make synthetic, recombinant triple-helical constructs that contain repeat segments of biologically relevant domains. Copies of a DNA fragment prepared by PCR from human type III collagen have been inserted in a co-linear contiguous fashion into the yeast expression vector YEpFlag-1, using sequential addition between selected restriction sites. Constructs containing 1, 2 and 3 repeats were designed to maintain the (Gly-X-Y) repeat, which is essential for the formation of an extended triple helix. All constructs gave expressed protein, with the best being the 3-repeat construct which was readily secreted. This material had the expected composition and N-terminal sequence. Incubation of the product at low temperature led to triple-helix formation, shown by reaction with a conformation dependent monoclonal antibody.
Peng
Yong Y
YY
CSIRO Molecular and Health Technologies, Bag 10, Clayton South, VIC 3169, Australia.
Werkmeister
Jerome A
JA
Vaughan
Paul R
PR
Ramshaw
John A M
JA
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D024061:Collagen Type III / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid
D024061:Collagen Type III