High level expression and facile purification of recombinant silk-elastin-like polymers in auto-induction shake flask cultures
Silk-elastin-like polymers (SELPs) are protein-based polymers which composition is based on the repetition of amino acid sequence motifs present in silk fibroin (GAGAGS) and in mammalian elastin (VPGVG). These copolymers have proven its importance for several biomedical applications and in the fabri...
| Main Author: | |
|---|---|
| Other Authors: | , , |
| Format: | conferencePoster |
| Language: | eng |
| Published: |
2011
|
| Online Access: | http://hdl.handle.net/1822/15565 |
| Country: | Portugal |
| Oai: | oai:repositorium.sdum.uminho.pt:1822/15565 |
| Summary: | Silk-elastin-like polymers (SELPs) are protein-based polymers which composition is based on the repetition of amino acid sequence motifs present in silk fibroin (GAGAGS) and in mammalian elastin (VPGVG). These copolymers have proven its importance for several biomedical applications and in the fabrication of micro- and nano-structures. Previously, SELPs were purified by immobilized metal affinity chromatography with volumetric productivities of 25-30 mg/l. With advances in recombinant DNA technology it is possible to design and produce tailored synthetic genes, creating multifunctional complex polymers, with total control on its composition and structure. In this work we report the construction of four new SELPs which composition is based on silk fibroin crystalline blocks and elastin-like thermoplastic (VPAVG) blocks. All the copolymers were constructed on the basis of different number of silk/elastin blocks and silk:elastin block ratios. The rationale is that by modifying the polymer structure it will exhibit different properties that will be further explored. Recombinant protein expression was achieved in Escherichia coli strain BL21(DE3) and purified by a chromatographic and non-chromatographic method. High levels of protein expression were obtained in bacterial cells cultured in terrific broth supplemented with lactose to allow auto-induction. The auto-induction cultures showed optimal recombinant expression in shake flask cultures with oxygen-limited conditions as determined by protein analysis in baffled and non-baffled flasks. Purification of recombinant copolymers was easily achieved by ammonium sulphate precipitation. The copolymers precipitated with 20% of ammonium sulphate with excellent recovery rates. A volumetric productivity of more than 150 mg/l was obtained for all the copolymers. |
|---|