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Synthetic proteins

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Synthetic Proteins

KARTHIKA VELUSAMY

PREETHA PALANISAMY

PRAGHADEESH MANIVANNAN

table=. =. |=.
p={color:#000;}.  

S.NO |=.
p={color:#000;}.  

CONTENTS |=.
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PAGE.NO | =. |=.
p={color:#000;}. 1.

|=. p={color:#000;}. Introduction |=. p={color:#000;}. 1 | =. |=. p={color:#000;}. 2. |=. p={color:#000;}. Synthetic biology |=. p={color:#000;}. 2 | =. |=/8. p={color:#000;}. 3.

3.1

3.2

3.3

3.4

3.5

3.6

3.7 |=.
p={color:#000;}. Chemical Synthesis of peptides |=.
p={color:#000;}. 3 | =. |=.
p={color:#000;}. Protein Synthesis by Peptide Ligation |=.
p={color:#000;}. 5 | =. |=.
p={color:#000;}. Peptide Ligation with Sulfur |=.
p={color:#000;}. 6 | =. |=.
p={color:#000;}. Native Chemical Ligation |=.
p={color:#000;}. 7 | =. |=.
p={color:#000;}. Expressed Protein Ligation |=.
p={color:#000;}. 8 | =. |=.
p={color:#000;}. Chemical Modification of Ligated Peptides |=.
p={color:#000;}. 9 | =. |=.
p={color:#000;}. Acyl-Initiated Capture |=.
p={color:#000;}. 10 | =. |=.
p={color:#000;}. Peptide Ligation With Selenium |=.
p={color:#000;}. 11 | =. |=/4.
p={color:#000;}. 4.

4.1

4.2

4.3 |=.
p={color:#000;}. General Strategies for Peptide Ligation |=.
p={color:#000;}. 12 | =. |=.
p={color:#000;}. Conformationally Assisted Ligation |=.
p={color:#000;}. 13 | =. |=.
p={color:#000;}. Removable Auxiliaries |=.
p={color:#000;}. 13 | =. |=.
p={color:#000;}. Staudinger Ligation |=.
p={color:#000;}. 15 | =. |=/13.
p={color:#000;}. 5.

5.1

5.2

5.3

5.3.1

5.3.2

5.3.3

5.4

5.5

5.6

5.7

5.8

5.9 |=.
p={color:#000;}. Biological synthesis of proteins |=.
p={color:#000;}. 17 | =. |=.
p={color:#000;}. Enzyme production |=.
p={color:#000;}. 19 | =. |=.
p={color:#000;}. Systems for producing recombinant proteins |=.
p={color:#000;}. 20 | =. |=.
p={color:#000;}. Bacteria |=.
p={color:#000;}. 21 | =. |=.
p={color:#000;}. E. coli |=.
p={color:#000;}. 21 | =. |=.
p={color:#000;}. Bacillus |=.
p={color:#000;}. 23 | =. |=.
p={color:#000;}. Other bacteria |=.
p={color:#000;}. 24 | =. |=.
p={color:#000;}. Yeasts |=.
p={color:#000;}. 25 | =. |=.
p={color:#000;}. Filamentous fungi (molds) |=.
p={color:#000;}. 26 | =. |=.
p={color:#000;}. Insect cells |=.
p={color:#000;}. 27 | =. |=.
p={color:#000;}. Mammalian cells |=.
p={color:#000;}. 28 | =. |=.
p={color:#000;}. Transgenic animals |=.
p={color:#000;}. 29 | =. |=.
p={color:#000;}. Transgenic plants |=.
p={color:#000;}. 31 | =. |=/6.
p={color:#000;}. 6.

6.1

6.2

6.3

6.4

6.5 |=.
p<{color:#000;background:#fff;}. Engineering proteins for environmental applications |=.
p={color:#000;background:transparent;}. 32 | =. |=.
p<{color:#000;background:#fff;}. Haloalkane dehalogenase |=.
p={color:#000;background:transparent;}. 32 | =. |=.
p<{color:#000;background:#fff;}. Methane monooxygenase |=.
p={color:#000;background:transparent;}. 33 | =. |=.
p<{color:#000;background:#fff;}. Cytochrome P450 |=.
p={color:#000;background:transparent;}. 34 | =. |=.
p<{color:#000;background:#fff;}. Ligninase |=.
p={color:#000;background:transparent;}. 34 | =. |=.
p<{color:#000;background:#fff;}. Aromatic dioxygenase |=.
p={color:#000;background:transparent;}. 35 | =. |=.
p={color:#000;}. 7.

=.
p={color:#000;}. Conclusions
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p={color:#000;}. 35

1. Introduction

Proteins are the most versatile among the various biological building blocks and mature field of protein engineering has lead to many industrial and biomedical applications. But the strength of proteins like their versatility, dynamics and interactions also complicates and hinders systems engineering. Therefore, the design of more sophisticated, multi-component protein systems appears to lag behind, in particular, when compared to the engineering of gene regulatory networks. Yet, synthetic biologists have started to tinker with the information flow through natural signalling networks or integrated protein switches. A successful strategy common to most of these experiments is their focus on modular interactions between protein domains or domains and peptide motifs. Such modular interaction swapping has rewired signaling in yeast, put mammalian cell morphology under the control of light, or increased the flux through a synthetic metabolic pathway.

For decades, protein engineering has been utilized to modify natural proteins and enzymes to meet the needs of different industrial applications. It is therefore a powerful tool in synthetic biology through the altering of protein properties to tailor protein “parts” to suit the requirements of any particular synthetic metabolic pathway or protein “devices” and systems. The study of natural proteins and the creation of nonnatural ones require the ability to access and manipulate proteins. The isolation of proteins from their natural source is often tedious, idiosyncratic, and impractical. In contrast, the production of proteins with recombinant DNA (rDNA) technology, either in a heterologous host or in vitro, can provide access to large quantities of protein and allow for the exchange of 1 of 20 common amino acid residues for another. However, aggregation often limits the yield of properly folded proteins produced with rDNA. Moreover, the restrictions of the genetic code severely limit the possible modifications.

The chemical synthesis and semisynthesis of proteins harbor the potential to overcome many of the disadvantages of current protein production methods (19,29,78). In particular, chemical synthesis using established solid-phase techniques are rapid to effect, easily automated, and facilitate purification. Chemical synthesis enables the facile incorporation of nonnatural functionality into proteins.

The desire to synthesize proteins is not new. On December 12, 1902, Emil Fischer delivered his Nobel Prize lecture in Stockholm, Sweden, saying in part:

Of the chemical aids in the living organism the ferments—mostly referred to nowadays as enzymes–-are so pre-eminent that they may justifiably be claimed to be involved in most of the chemical transformations in the living cell. The examination of the synthetic glucosides has shown that the action of the enzymes depends to a large extent on the geometrical structure of the molecule to be attacked, that the two must match like lock and key. Consequently, with their aid, the organism is capable of performing highly specific chemical transformations which can never be accomplished with the customary agents. To equal Nature here, the same means have to be applied, and I therefore foresee the day when physiological chemistry will not only make extensive use of the natural enzymes as agents, but when it will also prepare synthetic ferments for its purposes.

A century later, Fischer’s vision is becoming reality. Enzymes and other proteins not only are accessible targets for synthetic chemistry, but are poised to become dominant targets of the twenty-first century. Herein, we discuss current efforts toward preparing proteins synthetically, focusing on the development of powerful new methodologies for splicing peptide fragments in a convergent strategy for the total chemical synthesis of proteins.

2. Synthetic biology

Synthetic biology is a broad research area that combines biology and engineering to design and create biological systems with novel functions that are not found in nature. Although the term “synthetic biology” was first coined in 1980 (Hobom, 1980), the potential of the research field is only beginning to be realized (Liang et al., 2011). To date, advances in synthetic biology have facilitated production of biofuels (Nair and Zhao, 2010), specialty chemicals (Guo and Frost, 2004), pharmaceuticals (Chang and Keasling, 2006) and construction of novel quorum sensing mechanisms (Saeidi et al., 2011) using engineered biological systems. These achievements could lead the way to cost-effective synthesis of drugs, thus making them more affordable to Third World countries (Basu and Leong, 2011).

However, optimization of the engineered pathways to reach the required titer of the target molecules for industrial needs is no easy task. The highly regulated nature of biological systems often impedes the biosynthesis of the desired compounds by preventing overproduction of metabolites through feedback inhibition of enzymatic activities (Gerhart and Pardee, 1962). Additionally, foreign proteins may perform poorly in synthetic metabolic pathways and upset the intricate balance of the native metabolism, thus altering the metabolic flux and causing accumulation of toxic compounds which are detrimental to cell proliferation and product titer (Pitera et al., 2007). Moreover, if the target compounds do not exist in nature, the substrate specificities of natural enzymes may not be broad enough to convert the precursors to the products wanted. Owing to obstacles of such, production of compounds on an industrial scale using microorganisms with engineered metabolic pathways is still relatively rare due to sub-optimal yield from the unnatural biological systems.

For decades, protein engineering has been utilized to modify natural proteins and enzymes to meet the needs of different industrial applications. It is therefore a powerful tool in synthetic biology through the altering of protein properties to tailor protein “parts” to suit the requirements of any particular synthetic metabolic pathway.

3. Chemical synthesis of peptides

The chemical synthesis of proteins is now possible because of the prodigious advances in peptide synthesis that have occurred over the last century. Fischer’s 1901 synthesis of glycyl glycine is the first reported synthesis of a dipeptide and is also the first instance of the term “peptide” used to refer to a polymer of amino acids (Fischer and Fourneau, 1901). His 1907 synthesis of an octadecapeptide consisting of 15 glycine and 3 leucine residues was a remarkable achievement, despite his inability to control its amino acid sequence (Fischer, 1906).

An important advance in peptide synthesis was Bergmann & Zervas’ 1932 introduction of reversible protection for the α-amino group (Bergmann and Zervas, 1932). With the emergence of protecting group strategies, it became possible to synthesize small peptide hormones. For example, in 1953 du Vigneaud and coworkers (Du Vigneaud et al., 1953) reported a solution-phase synthesis of the octapeptide hormone oxytocin. Even though fifty years had passed since Fischer’s first synthesis of a peptide, these types of syntheses were still only accomplished with considerable effort (Merrifield, 1996).

The advent of solid-phase methods heralded a revolution for peptide synthesis (Merrifield, 1986). In 1963, Merrifield described the first solid-phase synthesis of a peptide, a tetrapeptide. He attached an amino acid to an insoluble support via its carboxyl group and then coupled the next amino acid, which had a protected amino group and an activated carboxyl group. The amino protecting group was removed, and the next amino acid was coupled in a similar manner.

Within a few years, Merrifield reported the development of an instrument for the automated synthesis of peptides. In short order, Gutte & Merrifield (1970) used this new strategy to achieve the first synthesis of an enzyme, ribonuclease A (RNase A), albeit in low overall yield. Concurrently, a team led by Hirschmann et al. 1969 reported the chemical synthesis of RNase S (which consists of residues 21–124 of RNase A) by solution-phase segment condensation reactions.

***

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Synthetic proteins

Synthetic biology is a broad research area that combines biology and engineering to design and create biological systems with novel functions that are not found in nature. Although the term “synthetic biology” was first coined in 1980, the potential of the research field is only beginning to be realized. To date, advances in synthetic biology have facilitated production of biofuels, specialty chemicals, pharmaceuticals and construction of novel quorum sensing mechanisms using engineered biological systems (Liang et al., 2011). For decades, protein engineering has been utilized to modify natural proteins and enzymes to meet the needs of different industrial applications. It is therefore a powerful tool in synthetic biology through the altering of protein properties to tailor protein “parts” to suit the requirements of any particular synthetic metabolic pathway. Transgenic microbes, animals and plants are tailored to express proteins for industrial applications (Foo et al., 2012). The chemical synthesis of proteins is alsio now possible because of the prodigious advances in peptide synthesis that have occurred over the last century. Fischer’s 1901 synthesis of glycyl glycine is the first reported synthesis of a dipeptide and is also the first instance of the term “peptide” used to refer to a polymer of amino acids. Peptide synthesis by chemical methods relies on peptide ligation by sulfur, native chemical ligation, expressed protein ligation, chemical modification of ligated peptides and so on (Nilsson et al., 2005). Compared to chemical synthesis, biological synthesis by genetic engineering is far better in economy wise and utility. These synthetic proteins are used as in various filed from food industry to automobile industries. Synthetic protein supplements, vaccines, antibodies, spider silk for fabrics, artificial meat and artificial blood carrier protein are few applications of synthetic proteins (Gomes et al., 2012).

  • ISBN: 9781311495136
  • Author: KARTHIKA VELUSAMY
  • Published: 2015-11-22 08:20:08
  • Words: 13961
Synthetic proteins Synthetic proteins