Introduction· a large number of functionally distinct proteins

Introduction·       Sortases: a brief introductionIn Gram-positive bacteria, the assembly of the pili and the attachment of virulence factors or proteins involved in heme iron acquisition and sporulation on the cell surface is performed by sortase enzymes. Sortases function as cysteine transpeptidases and join proteins bearing an appropriate sorting signal to strategically positioned amino groups on the cell wall1. During catalysis, sortases cleave a donor substrate containing the LPxTG (x = any amino acid) sorting motif under formation of an enzyme-bound thioester and ligate this intermediate to an acceptor protein containing an N-terminal glycine residue2.Approximately 60% of all sortase proteins can be divided into six different families of enzymes that share related amino acid sequences. Nevertheless, class A enzymes are the most studied group of sortases, and they appear to perform a housekeeping role in the cell as members of this group are capable of anchoring a large number of functionally distinct proteins to the cell wall1.

 ·       Sortase A: a potential drug target and a useful tool in protein chemistry Because of the role of sortase A (SrtA) in the attachment of virulence factors that allow bacteria to colonize and infect the host, this kind of enzymes have attracted significant interest as potential drug targets. The elimination of the srtA gene in several clinically significant bacteria is able to attenuate the virulence of these pathogens3. However, the usage of sortase inhibitors as an effective anti-bacterial therapy is still debated, as some studies indicate that these drugs are able to affect pathogenicity but not viability of bacteria4. By the other side, a recent investigation revealed that sortase inhibitors not only prevent the anchoring of virulence factors to the cell wall, but also lead to aberrant morphology, permeabilized membrane, increased sensitivity to anti-microbial peptides, and an inability to grow in the blood environment. Therefore, this evidence suggests that sortase inhibitors could be much better anti-bacterial agents than previously believed5. Besides the pharmacological inhibition of sortases to develop new anti-bacterial therapies, SrtA have been established as widely used tools in protein chemistry. The sortase-mediated ligation (SML) has found many applications including protein cyclization, solid-support immobilization, PEGylation, probing of protein-protein interactions, fluorescent tagging, generation of antibody drug conjugates, display and modification of phages and even in vivo ligation reactions6. Although Streptococcus aureus SrtA (Sa-SrtA) has been extensively studied and is the most commonly used enzyme for SML, several homologs of this enzyme have been identified.

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A recent report characterized the substrate specificity of different sortases from Staphylococci and Streptococci species. They found that Streptococci sortases displayed a more relaxed specificity for substrates than their Staphylococci counterparts2. Also, the structural and functional characterization of other StrA proteins has been performed in Lysteria monocytogenes7, Bacillus anthracis8, Actinomyces oris, and Corynebacterium diphtheria.Compared to other SrtA enzymes, the conjugation platform provided by Sa-SrtA has several strengths but also has some disadvantages.

The high specificity for the LPXTG motif and the wide range of oligo-glycine substrates which Sa-SrtA is able to catalyze, has positioned it as the favorite tool for SML6. Other great advantage of Sa-SrtA over Streptococcus pyogenes SrtA (Sp-SrtA) is the velocity at which this protein is able to perform the transpetidation reaction2. However, the main limitations for the industrial application of Sa-SrtA are its Ca2+ dependence for catalysis9 and its low thermostability. ·       Applications of a thermostable Staphylococcus aureus Sortase A (Sa-SrtA)  For many biotechnological purposes, it is desirable to use structurally and functionally stable proteins at higher temperatures. Chemical reactions are intrinsically faster at higher temperatures; therefore the usage of thermostable enzymes would lead to more efficient industrial processes. The enzyme biocatalysts required for industrial processes also need to withstand conditions that they are not usually exposed to within their natural environments such as high temperatures, extremes of pH, high salt, high substrate concentrations and the presence of organic solvents10. A previous study reported that Sa-SrtA remained its 50% of activity over a range of 20–60 °C, and that the optimal temperature for the activity of the enzyme is 35 °C11, which represent a very low value for its usage in industrial biotechnology.

Therefore, the development of a highly termostable Sa-SrtA is needed. ·       Experimental generation and screening of a thermostable Sa-SrtA Numerous studies on the origins of protein thermostability have suggested a variety of factors that can provide favorable stabilizing contributions, including the hydrophobic effect, improved molecular packing, van der Waals interactions, networks of hydrogen bonds and ionic bonds and optimized electrostatic interactions. It seems, however, that no dominating stabilization mechanism has evolved in proteins; rather, their thermostability results from a multitude of fine-tuned improvements of short-and long-range interactions12.

Several design approaches have already been developed for making proteins more thermally stable. In general, they involve random and rational approaches, which include directed evolution, computer design and site-directed mutagenesis. However, only some of these methods will be briefly described here. As mentioned, one of the most commonly used approaches for engineering proteins for thermostability is directed evolution, which attempts to accelerate natural evolution in a laboratory setting.

In this methodology, a number of genetic diversification techniques are used to generate libraries of gene variants. The resulting variants are then evaluated for improvement of a specific trait, those one that perform the best can be used for additional cycles of mutations until the protein variant with the desired property is obtained13. By the other side, if protein structure data are available, the B-factor iterative test (B-FIT) method can be used  to increase the thermal robustness of a protein of interest. This methodology considers the B-factors of a structure which are available from X ray data. B-factors account for the thermal motion of atoms.

A high average B-factor of a certain amino acid residue signifies a higher flexibility, as this residue has a low number of contacts to other amino acid residues. In order to obtain a more “rigid” and therefore a more thermostable protein structure, the highest B-factors are considered and subsequently target by iterative saturation mutagenesis14. Another approach is the consensus method which is based on the hypothesis that at a given position in an amino acid sequence alignment of homologous proteins, the respective consensus amino acid contributes more than average to the stability of the protein than the non consensus amino acids. Therefore, this computational-based methodology exploits the substitution of non consensus by consensus amino acids for the improvement of the thermostability of a given protein15. More recently, a method called Improved Configurational Entropy (ICE) has been used to create more thermally stable proteins. ICE is based on the redesign of proteins through optimization of their local structural entropy16. And finally, Rosetta Design has been also successfully applied to design proteins with increased stabilities. Rosetta Design is a computer program which has two main components, an energy function that ranks the relative fitness of various amino acid sequences for a given protein structure, and a search function for rapidly scanning sequence space17.

One of the main advantages of directed evolution over rational protein engineering is that it uses a process that resembles more closely natural protein evolution. Also, this approach is superior to iterative mutagenesis techniques, because it provides a mechanism to separate randomly beneficial and deleterious mutations. Nevertheless, the success of the directed evolution experiment is mainly determined by high throughput screening or selection methods to identify the mutants with the optimal combination of advantageous mutations18. Evaluation of individual protein variants is normally required in screening. It greatly reduces the chance of missing a desired mutant. However, the throughput is also reduced. By taking advantage of automation, high throughput screening methods can streamline traditional screening processes.

Most importantly, methods such as fluorescence resonance energy transfer (FRET) and fluorescence-activated cell sorting (FACS) can make the desired mutants easy to detect19. Therefore, only two of the most novel methods that take advantage of FRET and FACS and that allow the high throughput screening of SrtA activity will be discussed here. The FRET-based platform to measure SrtA activity is a highly sensitivity technique that utilize a FRET pair of fluorescent proteins, with the SrtA sorting motif LPETG attached to the carboxyl terminus of one of the fluorescent protein pairs (donor). By the other side, an oligoglycine peptide is fused to the amino terminus of the other fluorescent protein pair (acceptor). Upon SML, the FRET signal between donor and acceptor protein will be enhanced, therefore allowing the selection of the mutant active enzymes6 (Figure 1).   Figure 1.

A FRET-based platform is able to report SrtA activity. Figure was taken from Chen et al., 2016. The FACS-based platform to detect stabilizing mutations in proteins is called Cellular High-throughput Encapsulation Solubilization and Screening (CHESS). CHESS works by encapsulating single Escherichia coli (E.

coli) cells from a SrtA library in detergent-resistant polymers to form cell-like microcapsules. By physically encapsulating intracellular plasmids and the encoded proteins, these microcapsules maintain the gene:protein linkage, even upon exposure to harsh conditions such as membrane solubilization with detergents or high temperatures. Detergent or thermally stable mutant proteins are able to bind fluorescent labeled SrtA ligands, enabling the selection of microcapsules containing stable SrtA using FACS. Plasmids encoding stable SrtA variants can be extracted from the sorted microcapsules and can be applied to further rounds of CHESS, or sequences and used for biochemical and structural studies (Figure 2)20.  


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