Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP (original) (raw)
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Journal of Nano Research, 2009
Genetic engineering was used to produce an elastin-like polymer (ELP) with precise amino acid composition, sequence and length, resulting in the absolute control of MW and stereochemistry. A synthetic monomer DNA sequence encoding for (VPAVG) 20 , was used to build a library of concatemer genes with precise control on sequence and size. The higher molecular weight polymer with 220 repeats of VPAVG was biologically produced in Escherichia coli and purified by hot and cold centrifugation cycles, based on the reversible inverse temperature transition property of ELPs. The use of low cost carbon sources like lactose and glycerol for bacteria cells culture media was explored using Central Composite Design approach allowing optimization of fermentation conditions. Due to its self-assembling behaviour near 33 ºC stable spherical microparticles with a size ~ 1µm were obtained, redissolving when a strong undercooling is achieved. The polymer produced showed hysteresis behaviour with thermal absorbing/releasing components depending on the salt concentration of the polymer solution.
Biotechnology and Applied Biochemistry, 2005
Rapid progress has been made in the design and synthesis of oligomers and polymers that emulate the properties of natural proteins. Molecular bioengineering offers the chance to design and produce artificial polymeric proteins with tailored polymeric properties. The elastin-like polypeptides are a well-defined family of polymers with noteworthy characteristic based on the VPGVG repeated motif of bovine elastin. In the human homologue, the most regular sequence is represented by the repetition of the VAPGVG hexapeptidic motif. On the basis of this sequence, a synthetic gene has been designed, cloned and expressed in Escherichia coli to obtain artificial protein polymers. The rapid one-step in-frame cloning of any biologically active sequence can be achieved directly in the expression vector, allowing further improvement of the potential of the resulting product.
Biotechnology Progress, 1992
An elastomeric polypeptide was produced, with the sequence G-(VPGVG)19-VPGV, as a fusion to glutathione S-transferase using the vector pGEX-3X. The fusion protein was expressed to high levels in Escherichia coli as indicated by SDS-PAGE analysis of induced cells. The fusion protein was affinity purified and cleaved with protease factor Xa, and the elastomeric polypeptide was recovered to a high degree of purity as indicated by SDS-PAGE followed by staining with CuClz. The physical characterizations of carbon-13 and proton nuclear magnetic resonance and of the temperature profile for turbidity formation for the inverse temperature transition of hydrophobic folding and assembly attest to the successful microbial synthesis of the polypentapeptide of elastin. The results of these studies provide the initial progress toward achieving a more economical and practical means of producing material for elastic protein-based polymer research and applications.
Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers
World Journal of Microbiology and Biotechnology, 2014
Elastin-like polypeptides (ELP) are artificial, genetically encodable biopolymers, belonging to elastomeric proteins, which are widespread in a wide range of living organisms. They are composed of a repeating pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where the guest residue (Xaa) can be any naturally occurring amino acid except proline. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. This behavior depends greatly on the molecular weight, concentration of ELP in the solution and composition of the amino acids constituting ELPs. At a temperature below the inverse transition temperature (T t), ELPs are soluble, but insoluble when the temperature exceeds T t. Furthermore, this feature is retained even when ELP is fused to the protein of interest. These unique properties make ELP very useful for a wide variety of biomedical applications (e.g. protein purification, drug delivery etc.) and it can be expected that smart biopolymers will play a significant role in the development of most new materials and technologies. Here we present the structure and properties of thermally responsive elastin-like polypeptides with a particular emphasis on biomedical and biotechnological application.
We report a new strategy for the synthesis of genes encoding repetitive, protein-based polymers of specified sequence, chain length, and architecture. In this stepwise approach, which we term "recursive directional ligation" (RDL), short gene segments are seamlessly combined in tandem using recombinant DNA techniques. The resulting larger genes can then be recursively combined until a gene of a desired length is obtained. This approach is modular and can be used to combine genes encoding different polypeptide sequences. We used this method to synthesize three different libraries of elastin-like polypeptides (ELPs); each library encodes a unique ELP sequence with systematically varied molecular weights. We also combined two of these sequences to produce a block copolymer. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of these polypeptides provides an example of the importance of precise control over these parameters that is afforded by RDL.
Genetic engineering of structural protein polymers
Biotechnology Progress
Genetic and protein engineering are components of a new polymer chemistry that provide the tools for producing macromolecular polyamide copolymers of diversity and precision far beyond the current capabilities of synthetic polymer chemistry. The genetic machinery allows molecular control of chemical and physical chain properties. Nature utilizes this control to formulate protein polymers into materials with extraordinary mechanical properties, such as the strength and toughness of silk and the elasticity and resilience of mammalian elastin. The properties of these materials have been attributed to the presence of short repeating oligopeptide sequences contained in the proteins, fibroin, and elastin. We have produced homoblock protein polymers consisting exclusively of silk-like crystalline blocks and elastin-like flexible blocks. We have demonstrated that each homoblock polymer as produced by microbial fermentation exhibits measurable properties of crystallinity and elasticity. Additionally, we have produced alternating block copolymers of various amounts of silk-like and elastin-like blocks, ranging from a ratio of 1:4 to 2:1, respectively. The crystallinity of each copolymer varies with the amount of crystalline block interruptions. The production of fiber materials with custom-engineered mechanical properties is a potential outcome of this technology.
Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover 'smart' biopolymers that exhibit fully reversible thermally responsive behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a 'one-pot' parallel format. A rtificial repetitive polypeptides-also termed protein-polymers-derived from short peptide motifs found in elastin, collagen, silk and other structural proteins exhibit unique mechanical, structural and biological properties. These attributes have led to their application in biotechnology, tissue engineering, drug delivery and biosensing 1-5. Recombinant DNA technology is attractive for the synthesis of protein-polymers because it enables precise control of their length (number of repeats), composition and stereochemistry. This level of control is especially important for the in vivo applications of these biopolymers, because the polymer molecular weight controls their pharmacokinetics and biodistribution, whereas the amino acid sequence imparts biological activity to the biopolymer and affects their route, rate and mechanism of biodegradation. Recombinant DNA technology is also of interest for the synthesis of tandem repeats of naturally occurring peptides, as a strategy for the high-yield synthesis of peptide drugs and antigens 6-10. Furthermore, polymerization of peptide drugs with intervening protease cleavable sequences has the potential to improve their pharmacokinetics and drug efficacy 11,12. However, current methods for the polymerization of DNA suffer from one or more critical limitations: they (1) require numerous steps, (2) are difficult to run in parallel, and (3) do not provide tunable control over a range of molecular weights, all of which greatly limit the ability to simultaneously synthesize multiple variants with a range of repeat units and compositions. Motivated by these limitations, we report a rapid, one-step, high-throughput and robust method for the recombinant polymerization of 'monomer' DNA sequences with tunable control over the number of repeats. This method, which we term overlap extension rolling circle amplification (OERCA), uses rolling circle amplification (RCA) to produce linear repeats of a circularized gene, followed by thermally cycled overlap extension (OE) to yield a library of polymers of the monomer DNA, all in a single PCR reaction. Here we show the utility of OERCA and its advantages over existing methods by the synthesis of two classes of protein-polymers. First, we demonstrate the parallel synthesis of genes that encode elastin-like polypeptides (ELPs), a family of thermally responsive protein-polymers derived from a recurring VPGVG pentapeptide found in elastin 13. We used OERCA to rapidly synthesize nine different variants of ELPs, by substituting or inserting alanine residues along the VPGVG motif. These studies revealed an unexpected degree of sequence promiscuity in the parent peptide motif and yielded new families of 'smart' protein-polymers that exhibit fully reversible thermally responsive behaviour, which will provide a new set of stimulus responsive motifs for biomedical and biotechnological applications. In a second example, we use OERCA to rapidly synthesize protease-responsive polymers of glucagon-like peptide-1 (GLP-1) analogues, with intervening protease sites of variable potency for the optimization of in vivo pharmacokinetics and the release of GLP-1 from the polymer.
Improved Assembly of Multimeric Genes for the Biosynthetic Production of Protein Polymers
Biomacromolecules, 2002
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 -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 -sheet structure in aqueous solution.
2016
Citation: MacEwan, S.R., Hassouneh, W., Chilkoti, A. Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli. J. Vis. Exp. (88), e51583, doi:10.3791/51583 (2014). Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature p...