Understanding of the self- and co-assembly behavior of recombinant protein polymers : from design to implementation

M.D. Golinska

Research output: Thesisinternal PhD, WU

Abstract

A key part of the growing field of biomedical sciences deals with the development of new, controlled and biocompatible biomaterials. In this thesis we present results on the design, production, purification and characterization of stimuli responsive protein polymers that could ultimately be used in that field. Protein-polymers are composed of two or three blocks that are able to self- and co-assemble. An important theme in the thesis is to highlight the specific advantages of our new protein polymer for future biomedical applications.

We have used recombinant DNA techniques and expression in methylotrophic yeast Pichia pastorisfor protein production. DNAs encoding various polypeptide blocks were designed, produced using general molecular biology techniques and combined into synthetic genes for protein polymers. Synthetic genes were cloned into P. pastorisexpression vector pPIC9 that integrates into the yeast genome. Yields were high, typically reaching gram-per-liter (of medium).

In Chapter 2we study dual-stimuli (pH, temperature) responsive silk-elastin-like protein polymers (SELPs). These polymers were designed to self- and co-assembly, controlled by both pH and temperature. The first protein is a diblock S24E40composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats. The other protein is a triblock S12C4E40, in which the Sand Eblocks are separated by a random coil block (C4) that serves as an inert ‘spacer’.

A C2SHSHC2protein polymer, which consists of a pH responsive, positively charged silk-like middle block SH, flanked by two random coil collagen-like blocks C2was studied in Chapter 3.

For this protein have studied fibril formation and gelling properties at pH values close to neutral, that are crucial for biomedical applications. We find that at physiological pH, these proteins form self-healing physical gels that fulfill many requirements for use in biomedical applications.


 

 

In Chapter 4we test the influence of enzymatic cross-linking on elasticity andmechanical properties ofhydrogels that include collagen-like domains, using microbial transglutaminase (mTGase) as an enzymatic crosslinker that catalyzes the coupling of glutamines to lysines. We show that even though the collagen-like blocks are not particularly good substrates for the mTGase, the few cross-links that are made have a strong effect on the physical properties of the protein-polymer hydrogels. For silk-collagen fiber gels, the elastic moduli can be increased by a factor of five, and for thermosensitive collagen hydrogels, the enzymatic cross-linking induces qualitatively new behavior, namely shape-memory of hydrogels.

Finally, we study the co-assembly of very asymmetric diblock copolymers with oppositely charged sodium poly(acrylic acid) (NaPAA) with a range of molar masses (Chapter 5). This asymmetric diblock consists of a cationic block of 12 lysines connected to a long (400 amino acid) collagen-like block with a net charge that is nearly zero. For shorter Na-PAA chains, spherical complex coacervates micelles are formed, as have been studied before in our lab. But, for long Na-PAA chains a new self-assembled structure is found: a single (Na-PAA) chain pearl-necklace of complex-coacervate micelles.

The general discussion of the thesis in Chapter 6, focuses on recombinant and natural hydrogels as biomaterials. We point out the specific advantages of recombinant proteins and also indicate where these still need to be improved in order to be used in biomedical applications. Finally, we make some suggestions for further research in this area.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Cohen Stuart, Martien, Promotor
  • de Vries, Renko, Co-promotor
  • de Wolf, Frits, Co-promotor
Award date7 Jan 2014
Place of PublicationWageningen
Publisher
Print ISBNs9789461738134
Publication statusPublished - 2014

Fingerprint

Recombinant Proteins
Polymers
Hydrogels
Silk
Collagen
Proteins
Transglutaminases
Elastin
carbopol 940
Micelles
Biocompatible Materials
Yeast
Lysine
Genes
Gels
Molecular biology
Recombinant DNA
Molar mass
Glutamine
Shape memory effect

Keywords

  • biopolymers
  • proteins
  • self assembly
  • recombinant proteins
  • elastin
  • collagen
  • enzymatic cross-linking
  • glutaminase

Cite this

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title = "Understanding of the self- and co-assembly behavior of recombinant protein polymers : from design to implementation",
abstract = "A key part of the growing field of biomedical sciences deals with the development of new, controlled and biocompatible biomaterials. In this thesis we present results on the design, production, purification and characterization of stimuli responsive protein polymers that could ultimately be used in that field. Protein-polymers are composed of two or three blocks that are able to self- and co-assemble. An important theme in the thesis is to highlight the specific advantages of our new protein polymer for future biomedical applications. We have used recombinant DNA techniques and expression in methylotrophic yeast Pichia pastorisfor protein production. DNAs encoding various polypeptide blocks were designed, produced using general molecular biology techniques and combined into synthetic genes for protein polymers. Synthetic genes were cloned into P. pastorisexpression vector pPIC9 that integrates into the yeast genome. Yields were high, typically reaching gram-per-liter (of medium). In Chapter 2we study dual-stimuli (pH, temperature) responsive silk-elastin-like protein polymers (SELPs). These polymers were designed to self- and co-assembly, controlled by both pH and temperature. The first protein is a diblock S24E40composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats. The other protein is a triblock S12C4E40, in which the Sand Eblocks are separated by a random coil block (C4) that serves as an inert ‘spacer’. A C2SHSHC2protein polymer, which consists of a pH responsive, positively charged silk-like middle block SH, flanked by two random coil collagen-like blocks C2was studied in Chapter 3. For this protein have studied fibril formation and gelling properties at pH values close to neutral, that are crucial for biomedical applications. We find that at physiological pH, these proteins form self-healing physical gels that fulfill many requirements for use in biomedical applications.     In Chapter 4we test the influence of enzymatic cross-linking on elasticity andmechanical properties ofhydrogels that include collagen-like domains, using microbial transglutaminase (mTGase) as an enzymatic crosslinker that catalyzes the coupling of glutamines to lysines. We show that even though the collagen-like blocks are not particularly good substrates for the mTGase, the few cross-links that are made have a strong effect on the physical properties of the protein-polymer hydrogels. For silk-collagen fiber gels, the elastic moduli can be increased by a factor of five, and for thermosensitive collagen hydrogels, the enzymatic cross-linking induces qualitatively new behavior, namely shape-memory of hydrogels. Finally, we study the co-assembly of very asymmetric diblock copolymers with oppositely charged sodium poly(acrylic acid) (NaPAA) with a range of molar masses (Chapter 5). This asymmetric diblock consists of a cationic block of 12 lysines connected to a long (400 amino acid) collagen-like block with a net charge that is nearly zero. For shorter Na-PAA chains, spherical complex coacervates micelles are formed, as have been studied before in our lab. But, for long Na-PAA chains a new self-assembled structure is found: a single (Na-PAA) chain pearl-necklace of complex-coacervate micelles. The general discussion of the thesis in Chapter 6, focuses on recombinant and natural hydrogels as biomaterials. We point out the specific advantages of recombinant proteins and also indicate where these still need to be improved in order to be used in biomedical applications. Finally, we make some suggestions for further research in this area.",
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year = "2014",
language = "English",
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Golinska, MD 2014, 'Understanding of the self- and co-assembly behavior of recombinant protein polymers : from design to implementation', Doctor of Philosophy, Wageningen University, Wageningen.

Understanding of the self- and co-assembly behavior of recombinant protein polymers : from design to implementation. / Golinska, M.D.

Wageningen : Wageningen University, 2014. 165 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Understanding of the self- and co-assembly behavior of recombinant protein polymers : from design to implementation

AU - Golinska, M.D.

N1 - WU thesis 5664

PY - 2014

Y1 - 2014

N2 - A key part of the growing field of biomedical sciences deals with the development of new, controlled and biocompatible biomaterials. In this thesis we present results on the design, production, purification and characterization of stimuli responsive protein polymers that could ultimately be used in that field. Protein-polymers are composed of two or three blocks that are able to self- and co-assemble. An important theme in the thesis is to highlight the specific advantages of our new protein polymer for future biomedical applications. We have used recombinant DNA techniques and expression in methylotrophic yeast Pichia pastorisfor protein production. DNAs encoding various polypeptide blocks were designed, produced using general molecular biology techniques and combined into synthetic genes for protein polymers. Synthetic genes were cloned into P. pastorisexpression vector pPIC9 that integrates into the yeast genome. Yields were high, typically reaching gram-per-liter (of medium). In Chapter 2we study dual-stimuli (pH, temperature) responsive silk-elastin-like protein polymers (SELPs). These polymers were designed to self- and co-assembly, controlled by both pH and temperature. The first protein is a diblock S24E40composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats. The other protein is a triblock S12C4E40, in which the Sand Eblocks are separated by a random coil block (C4) that serves as an inert ‘spacer’. A C2SHSHC2protein polymer, which consists of a pH responsive, positively charged silk-like middle block SH, flanked by two random coil collagen-like blocks C2was studied in Chapter 3. For this protein have studied fibril formation and gelling properties at pH values close to neutral, that are crucial for biomedical applications. We find that at physiological pH, these proteins form self-healing physical gels that fulfill many requirements for use in biomedical applications.     In Chapter 4we test the influence of enzymatic cross-linking on elasticity andmechanical properties ofhydrogels that include collagen-like domains, using microbial transglutaminase (mTGase) as an enzymatic crosslinker that catalyzes the coupling of glutamines to lysines. We show that even though the collagen-like blocks are not particularly good substrates for the mTGase, the few cross-links that are made have a strong effect on the physical properties of the protein-polymer hydrogels. For silk-collagen fiber gels, the elastic moduli can be increased by a factor of five, and for thermosensitive collagen hydrogels, the enzymatic cross-linking induces qualitatively new behavior, namely shape-memory of hydrogels. Finally, we study the co-assembly of very asymmetric diblock copolymers with oppositely charged sodium poly(acrylic acid) (NaPAA) with a range of molar masses (Chapter 5). This asymmetric diblock consists of a cationic block of 12 lysines connected to a long (400 amino acid) collagen-like block with a net charge that is nearly zero. For shorter Na-PAA chains, spherical complex coacervates micelles are formed, as have been studied before in our lab. But, for long Na-PAA chains a new self-assembled structure is found: a single (Na-PAA) chain pearl-necklace of complex-coacervate micelles. The general discussion of the thesis in Chapter 6, focuses on recombinant and natural hydrogels as biomaterials. We point out the specific advantages of recombinant proteins and also indicate where these still need to be improved in order to be used in biomedical applications. Finally, we make some suggestions for further research in this area.

AB - A key part of the growing field of biomedical sciences deals with the development of new, controlled and biocompatible biomaterials. In this thesis we present results on the design, production, purification and characterization of stimuli responsive protein polymers that could ultimately be used in that field. Protein-polymers are composed of two or three blocks that are able to self- and co-assemble. An important theme in the thesis is to highlight the specific advantages of our new protein polymer for future biomedical applications. We have used recombinant DNA techniques and expression in methylotrophic yeast Pichia pastorisfor protein production. DNAs encoding various polypeptide blocks were designed, produced using general molecular biology techniques and combined into synthetic genes for protein polymers. Synthetic genes were cloned into P. pastorisexpression vector pPIC9 that integrates into the yeast genome. Yields were high, typically reaching gram-per-liter (of medium). In Chapter 2we study dual-stimuli (pH, temperature) responsive silk-elastin-like protein polymers (SELPs). These polymers were designed to self- and co-assembly, controlled by both pH and temperature. The first protein is a diblock S24E40composed of 24 silk-like (S) repeats and 40 elastin-like (E) repeats. The other protein is a triblock S12C4E40, in which the Sand Eblocks are separated by a random coil block (C4) that serves as an inert ‘spacer’. A C2SHSHC2protein polymer, which consists of a pH responsive, positively charged silk-like middle block SH, flanked by two random coil collagen-like blocks C2was studied in Chapter 3. For this protein have studied fibril formation and gelling properties at pH values close to neutral, that are crucial for biomedical applications. We find that at physiological pH, these proteins form self-healing physical gels that fulfill many requirements for use in biomedical applications.     In Chapter 4we test the influence of enzymatic cross-linking on elasticity andmechanical properties ofhydrogels that include collagen-like domains, using microbial transglutaminase (mTGase) as an enzymatic crosslinker that catalyzes the coupling of glutamines to lysines. We show that even though the collagen-like blocks are not particularly good substrates for the mTGase, the few cross-links that are made have a strong effect on the physical properties of the protein-polymer hydrogels. For silk-collagen fiber gels, the elastic moduli can be increased by a factor of five, and for thermosensitive collagen hydrogels, the enzymatic cross-linking induces qualitatively new behavior, namely shape-memory of hydrogels. Finally, we study the co-assembly of very asymmetric diblock copolymers with oppositely charged sodium poly(acrylic acid) (NaPAA) with a range of molar masses (Chapter 5). This asymmetric diblock consists of a cationic block of 12 lysines connected to a long (400 amino acid) collagen-like block with a net charge that is nearly zero. For shorter Na-PAA chains, spherical complex coacervates micelles are formed, as have been studied before in our lab. But, for long Na-PAA chains a new self-assembled structure is found: a single (Na-PAA) chain pearl-necklace of complex-coacervate micelles. The general discussion of the thesis in Chapter 6, focuses on recombinant and natural hydrogels as biomaterials. We point out the specific advantages of recombinant proteins and also indicate where these still need to be improved in order to be used in biomedical applications. Finally, we make some suggestions for further research in this area.

KW - biopolymeren

KW - eiwitten

KW - zelf-assemblage

KW - recombinant eiwitten

KW - elastine

KW - collageen

KW - enzymatische cross-linking

KW - glutaminase

KW - biopolymers

KW - proteins

KW - self assembly

KW - recombinant proteins

KW - elastin

KW - collagen

KW - enzymatic cross-linking

KW - glutaminase

M3 - internal PhD, WU

SN - 9789461738134

PB - Wageningen University

CY - Wageningen

ER -