Improved assembly of multimeric genes for the biosynthetic production of protein polymers.
We report a general method for the construction of highly repetitive synthetic genes and their use in the biosynthetic production of artificial protein polymers. Through the application of improved recombinant DNA techniques and high-throughput screening methods, we have developed a facile approach to rapid gene assembly and cloning which is widely applicable in the biosynthesis of novel protein polymers. Using this technique, synthetic genes encoding tandem repeats of the beta-sheet forming amino acid sequence AEAEAKAK were constructed and subsequently cloned into a bacterial expression host for inducible protein production. A 17-kDa fusion protein, poly-EAK9, was isolated from Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. The amino acid sequence and molecular weight were confirmed by amino acid analysis, N-terminal sequencing, and MALDI-TOF mass spectrometry. Circular dichroism studies on the artificial protein poly-EAK9 demonstrate the formation of a beta-sheet structure in aqueous solution.
Goeden-Wood
Nichole L
NL
Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 94720-1462, USA.
Conticello
Vincent P
VP
Muller
Susan J
SJ
Keasling
Jay D
JD
D000595:Amino Acid Sequence; D001704:Biopolymers / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005813:Genes, Synthetic / Q000235:genetics*; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000235:genetics; D020080:Tandem Repeat Sequences
D001704:Biopolymers; D011506:Proteins