Expression and purification of a spider silk protein: a new strategy for producing repetitive proteins.
Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced in Escherichia coli at levels up to 10 mg/g wet wt of cells although yields of 1-2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers.
Lewis
R V
RV
Department of Molecular Biology, University of Wyoming, Laramie 82071-3944, USA. silk@uwyo.edu
Hinman
M
M
Kothakota
S
S
Fournier
M J
MJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001345:Autoradiography; D001483:Base Sequence; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005349:Fibroins; D015870:Gene Expression; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D013862:Thiogalactosides / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D013862:Thiogalactosides; C076815:spidroin 2; D005349:Fibroins; D014357:Trypsin