"

From 2014.igem.org

Revision as of 10:15, 13 October 2014 (view source) MaxW (Talk | contribs) (Created page with "Hier schreibt Max") ← Older edit		Revision as of 10:42, 13 October 2014 (view source) MaxW (Talk | contribs) Newer edit →
Line 1:		Line 1:
-	Hier schreibt Max	+	==Circularization==
		+
		+	===Introduction===
		+	Proteins normally appear in nature with two ends, the N-terminal amino and the C-terminal carboxylic groups. By linking those two ends, it is possible circularize these proteins and introduce interesting properties. Circular proteins can be superior to linear proteins in terms of thermostability [[#References|[1][2][3]]], stability against chemical denaturation [[#References|[4]]] and exopeptidase resistance [[#References|[2][4]]], while the biological function of he linear counterpart is conserved [[#References|[5]]].
		+	Improving thermostability of enzymes is an essential aspect for industrial applications [[#References|[5]]]. Indeed, in many applications, those enzymes are required to work at temperatures that would normally cause denaturation [[#References|[6]]]. Other recombinant proteins, for example enzymes used in laboratories, are required to be as stable as possible at moderate temperatures [[#References|[6]]]. Moreover, a circular backbone can improve in vivo stability of therapeutical proteins and peptides [[#References|[5]]].
		+	Our circularization tools allow easy expression of circular proteins in e. coli. Choose a protein you want to circularize and use our [toolbox guide]!
		+
		+	===Circular proteins in nature===
		+	Due to their extraordinary features, circular proteins have emerged independently in different organisms. Those proteins are rather small (<79 amino acids) and most often them are additionally stabilized by disulfide bonds [[#References|[7]]].
		+	A pharmaceutically active cyclic peptide from a plant, katala B1, has been used by African women to accelerate childbirth and was obtained by boiling the dry plant leaves, demonstrating its considerable thermostability [[#References|[8]]]. This cyclic peptide belongs to the large family of cyclotides, which might have a great potential in drug design [[#References|[7]]].
		+	Most of the circular peptides found in nature have a defense function. Rhesus θ-defensin-1, found in rhesus macaque leukocytes, is a highly potent antibiotic peptide [[#References|[9]]]. Cyclosporine, a small circular peptide from fungi, is able to suppress the human immune system and thus important for transplant surgery [[#References|[7]]].
		+	Importantly, naturally occurring circularization of more complex and larger proteins have not yet been described. Considering the benefit that one can expect from improved protein stability, we anticipated that developing tools to circularize proteins would be of great help for the scientific and industrial communities.
		+
		+	===What has been done?===
		+	For that part, you may want to cite the review: Biological synthesis of circular polypeptides, Aboye and Camarero, JBC, 2012
		+	Using [split inteins] or [sortase], it is possible to cirularize linear proteins. The most important proteins that were circularized using split inteins are dihydrofolate reductase [[#References|[1]]] and GFP [[#References|[4]]]. Those proteins are rather small and their termini are in close proximity. Both circular dihydrofolate reductase and GFP showed enhanced resistance against proteases and denaturation.
		+	The largest protein circular protein yet is maltose-binding protein (MBP) (395 aa) from E. coli [[#References|[10]]]. However, stability was not tested in this case.
		+	A GFP variant was also circularized in vitro using sortase. In this case, faster recovery of fluorescence after denaturation was observed in comparison with a linear control [[#References|[11]]].
		+
		+
		+	===What have we done?===
		+	Our interest for protein circularization originated from our ambitious idea to create a thermostable DNA methyltransferase that could be included in a polymerase chain reaction (PCR) to transfer the methylation state of the original DNA to the product. Knowing the importance of DNA methylation in gene expression regulation, such a PCR assay would be a revolution for epigenetics. In practice, we aimed at circularizing DNA (cytosine-5)-methyltransferase 1 (DNMT1) [DNMT1-Artikel]. If DNMT1 and the cofactor S-adenosyl methionine (SAM) could simply be added to a conventional PCR reaction, DNA could be amplified without losing methylation..
		+	Consequently, we planned to circularize DNMT1 in order to improve its thermostability as much as possible. We used GFP to test both split intein and sortase circularization. We successfully circularized GFP in vivo using a preliminary split intein-based circularization construct [proof].
		+	However, even the most truncated yet functional form of DNMT1 (872 aa) is much larger than GFP or MBP and its termini are far away from each other. Therefore, it is necessary to put a large linker between the termini. A long flexible linker would probably not be able to significantly enhance thermostability, since the termini would not be locked in one position.
		+	So we came up with the idea to design rigid linkers with angles [verweis auf nils‘ zeug] and developed a software to generate linkers for any protein [nochmal verweis auf nils zeug]. We circularized lambda lysozyme in order to test and calibrate our linker model [verweis lalys]. Circular lambda lysozyme with 9 different linkers was screened for thermostability.
		+
		+	===What could be done?===
		+
		+
		+	===How does it work?===
		+	 our constructs, warum super und notwendig
		+	Wie wird thermostabilität erreicht?
		+
		+	===References===
		+
		+	[1] Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. 96, 13638–13643 (1999).
		+
		+	[2] Iwai, H. & Plückthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166–72 (1999).
		+
		+	[3] Flory, J. & Yol, S. Theory of Elastic Mechanisms in Fibrous Proteins. 715, 5222–5235 (1956)
		+
		+	[4] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548–54 (2001).
		+
		+	[5] Trabi, M. & Craik, D. J. Circular proteins--no end in sight. Trends Biochem. Sci. 27, 132–8 (2002).
		+
		+	[6] Biology, S. Factors increasing protein thermostability. 1–41 (2002).
		+
		+	[7] Craik, D. J. Chemistry. Seamless proteins tie up their loose ends. Science 311, 1563–4 (2006).
		+
		+	[8] A, P. K. B. et al. Elucidation of the Primary and Three-Dimensional Structure of the Uterotonic. 4147–4158 (1995).
		+
		+	[9] Tang, Y. A Cyclic Antimicrobial Peptide Produced in Primate Leukocytes by the Ligation of Two Truncated -Defensins. Science (80-. ). 286, 498–502 (1999).
		+
		+	[10] Evans, T. C. Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene of Synechocystis Species PCC6803. J. Biol. Chem. 275, 9091–9094 (2000).
		+
		+	[11] Antos, J. M. et al. A straight path to circular proteins. J. Biol. Chem. 284, 16028–36 (2009).

---

Revision as of 10:42, 13 October 2014

Contents 1 Circularization 1.1 Introduction 1.2 Circular proteins in nature 1.3 What has been done? 1.4 What have we done? 1.5 What could be done? 1.6 How does it work? 1.7 References

Circularization

Introduction

Proteins normally appear in nature with two ends, the N-terminal amino and the C-terminal carboxylic groups. By linking those two ends, it is possible circularize these proteins and introduce interesting properties. Circular proteins can be superior to linear proteins in terms of thermostability [1][2][3], stability against chemical denaturation [4] and exopeptidase resistance [2][4], while the biological function of he linear counterpart is conserved [5]. Improving thermostability of enzymes is an essential aspect for industrial applications [5]. Indeed, in many applications, those enzymes are required to work at temperatures that would normally cause denaturation [6]. Other recombinant proteins, for example enzymes used in laboratories, are required to be as stable as possible at moderate temperatures [6]. Moreover, a circular backbone can improve in vivo stability of therapeutical proteins and peptides [5]. Our circularization tools allow easy expression of circular proteins in e. coli. Choose a protein you want to circularize and use our [toolbox guide]!

Circular proteins in nature

Due to their extraordinary features, circular proteins have emerged independently in different organisms. Those proteins are rather small (<79 amino acids) and most often them are additionally stabilized by disulfide bonds [7]. A pharmaceutically active cyclic peptide from a plant, katala B1, has been used by African women to accelerate childbirth and was obtained by boiling the dry plant leaves, demonstrating its considerable thermostability [8]. This cyclic peptide belongs to the large family of cyclotides, which might have a great potential in drug design [7]. Most of the circular peptides found in nature have a defense function. Rhesus θ-defensin-1, found in rhesus macaque leukocytes, is a highly potent antibiotic peptide [9]. Cyclosporine, a small circular peptide from fungi, is able to suppress the human immune system and thus important for transplant surgery [7]. Importantly, naturally occurring circularization of more complex and larger proteins have not yet been described. Considering the benefit that one can expect from improved protein stability, we anticipated that developing tools to circularize proteins would be of great help for the scientific and industrial communities.

What has been done?

For that part, you may want to cite the review: Biological synthesis of circular polypeptides, Aboye and Camarero, JBC, 2012 Using [split inteins] or [sortase], it is possible to cirularize linear proteins. The most important proteins that were circularized using split inteins are dihydrofolate reductase [1] and GFP [4]. Those proteins are rather small and their termini are in close proximity. Both circular dihydrofolate reductase and GFP showed enhanced resistance against proteases and denaturation. The largest protein circular protein yet is maltose-binding protein (MBP) (395 aa) from E. coli [10]. However, stability was not tested in this case. A GFP variant was also circularized in vitro using sortase. In this case, faster recovery of fluorescence after denaturation was observed in comparison with a linear control [11].

What have we done?

Our interest for protein circularization originated from our ambitious idea to create a thermostable DNA methyltransferase that could be included in a polymerase chain reaction (PCR) to transfer the methylation state of the original DNA to the product. Knowing the importance of DNA methylation in gene expression regulation, such a PCR assay would be a revolution for epigenetics. In practice, we aimed at circularizing DNA (cytosine-5)-methyltransferase 1 (DNMT1) [DNMT1-Artikel]. If DNMT1 and the cofactor S-adenosyl methionine (SAM) could simply be added to a conventional PCR reaction, DNA could be amplified without losing methylation.. Consequently, we planned to circularize DNMT1 in order to improve its thermostability as much as possible. We used GFP to test both split intein and sortase circularization. We successfully circularized GFP in vivo using a preliminary split intein-based circularization construct [proof]. However, even the most truncated yet functional form of DNMT1 (872 aa) is much larger than GFP or MBP and its termini are far away from each other. Therefore, it is necessary to put a large linker between the termini. A long flexible linker would probably not be able to significantly enhance thermostability, since the termini would not be locked in one position. So we came up with the idea to design rigid linkers with angles [verweis auf nils‘ zeug] and developed a software to generate linkers for any protein [nochmal verweis auf nils zeug]. We circularized lambda lysozyme in order to test and calibrate our linker model [verweis lalys]. Circular lambda lysozyme with 9 different linkers was screened for thermostability.

What could be done?

How does it work?

 our constructs, warum super und notwendig Wie wird thermostabilität erreicht?

References

[1] Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. 96, 13638–13643 (1999).

[2] Iwai, H. & Plückthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166–72 (1999).

[3] Flory, J. & Yol, S. Theory of Elastic Mechanisms in Fibrous Proteins. 715, 5222–5235 (1956)

[4] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548–54 (2001).

[5] Trabi, M. & Craik, D. J. Circular proteins--no end in sight. Trends Biochem. Sci. 27, 132–8 (2002).

[6] Biology, S. Factors increasing protein thermostability. 1–41 (2002).

[7] Craik, D. J. Chemistry. Seamless proteins tie up their loose ends. Science 311, 1563–4 (2006).

[8] A, P. K. B. et al. Elucidation of the Primary and Three-Dimensional Structure of the Uterotonic. 4147–4158 (1995).

[9] Tang, Y. A Cyclic Antimicrobial Peptide Produced in Primate Leukocytes by the Ligation of Two Truncated -Defensins. Science (80-. ). 286, 498–502 (1999).

[10] Evans, T. C. Protein trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene of Synechocystis Species PCC6803. J. Biol. Chem. 275, 9091–9094 (2000).

[11] Antos, J. M. et al. A straight path to circular proteins. J. Biol. Chem. 284, 16028–36 (2009).