Sterilized recombinant spider silk fibers of low pyrogenicity.
We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli , the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.
Hedhammar
My
M
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, SE-751 23 Uppsala, Sweden. my.hedhammar@afb.slu.se
Bramfeldt
Hanna
H
Baris
Teodora
T
Widhe
Mona
M
Askarieh
Glareh
G
Nordling
Kerstin
K
Aulock
Sonja von
Sv
Johansson
Jan
J
D000818:Animals; D002478:Cells, Cultured; D020405:Dermis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005349:Fibroins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D008070:Lipopolysaccharides / Q000494:pharmacology; D015202:Protein Engineering; D011749:Pyrogens / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013112:Spiders / Q000737:chemistry*; D013242:Sterilization; D013718:Tensile Strength
D008070:Lipopolysaccharides; D011749:Pyrogens; D011994:Recombinant Proteins; C086087:spidroin 1; D005349:Fibroins