Part:BBa_K2136002
Lysostaphin
Lysostaphin is a 27 kDa-zinc metalloproteinase (EC 3.4.24.75) produced by Staphylococcus simulans. This enzyme hydrolyses glycine/glycine bonds in the pentaglycine interpeptide that maintain the stability of peptidoglycans in staphylococcal cell wall (Surovtsev 1).
Usage
In spite of recent advances in medical care, data reveals that mortality rate among burned patients remains higher depending on the extent of affected area (Pavoni 2). These death rates could also be driven by prolonged antibiotic courses, which related to immunocompetence decrease. Consequently, leading patient to a vulnerability state of the skin to microorganisms (Baker 3), besides the per se lost of the first line of antimicrobial defense. Given the fact that hospital acquired infections is a growing concern in public health alongside the development of resistant strains, burned individuals are at the risk top risk among patients (Leseva 4).
Biology
Lysostaphin is encoded by a three-modular gene that encompasses a self-cleavage peptide, a propetide and the self-coding sequence of lysostaphin. After protein processing, mature lysostaphin (246 aminoacids) is released and exhibits a triple-enzymatic activity: glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine. Consequently, it rapidly lyses actively growing and non-dividing cells including staphylococci in biofilms and, due to its specificity, it could have high potential in the treatment of antibiotic-resistant staphylococcal infections (Kumar 5).
Perspectives
This protein with antibiotic properties seems as a promissory candidate for medical purposes that combined with novel chimeric spider silk as polymeric matrix could become an interesting approach to address nosocomial infections by resistant strains.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 505
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
It’s important to take into account that this sequence encompasses only mature lysostaphin.
As our project relies on the advantage of Chlamydomonas reinhardtii as a proper chassis for high GC-content genes, our team adapted mature coding sequence of lysostaphin and codon optimized for microalgae. Notice that stop codon was removed from original sequence.
Cloning into pSB1C3 vector
For further details, explore our complete experimental part!
Analytical digestion
After several trials of transformation in DH-5α cells, plasmid bearing lysostaphin sequence was obtained. Analytical digestion was normally performed with about 500ng of plasmid.
<img src="" style="margin-bottom:20px; width="20px" margin-top:0px;" />
Figure 1. Analytical digestion pSB1C3 + Lysostaphin
PCR confirmation
From plasmid backbone, PCR reaction was performed in order to identify the insert. Either our home-made X7 and Q5 polymerase worked perfectly.
<img src="" style="margin-bottom:20px; width="20px" margin-top:0px;" />
Figure 2.PCR from pSB1C3 backbone
Sequencing
Through the last months, lysostaphin feed our hopes and became one of our favorite parts. In order to confirm that BBa_K2136002 was cloned properly, sequencing was performed under standard protocol provided by local service.
Sequencing with forward primer
<img src="" style="margin-bottom:20px; width="20px" margin-top:0px;" />
Figure 3.</b>Sequencing reaction for forward primer
Sequencing with reverse primer
<img src="" style="margin-bottom:20px; width="20px" margin-top:0px;" />
Figure 4.</b>Sequencing reaction for reverse primer
For further details of sequencing data, check here!
References
Baker, Christopher C., Carol L. Miller, and DONALD D. TRUNKEY. "Predicting fatal sepsis in burn patients." Journal of Trauma and Acute Care Surgery 19.9 (1979): 641-648.
Hojckova, Katarina, Matej Stano, and Lubos Klucar. "phiBIOTICS: catalogue of therapeutic enzybiotics, relevant research studies and practical applications." BMC microbiology 13.1 (2013): 1.
Leseva, M., et al. "Nosocomial infections in burn patients: etiology, antimicrobial resistance, means to control." Ann Burns Fire Disasters 26.1 (2013): 5-11.
Kumar, Jaspal K. "Lysostaphin: an antistaphylococcal agent." Applied microbiology and biotechnology 80.4 (2008): 555-561.
Hardy J, Römer L, Scheibel T (2008) Polymeric materials based on silk proteins. Polymer 49 (20): 4309-4327. DOI: 10.1016/j.polymer.2008.08.006
Kluge J, Rabotyagova O, Leisk G, Kaplan D (2008) Spider silks and their applications. Trends in Biotechnology 26 (5): 244-251. DOI: 10.1016/j.tibtech.2008.02.006
Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g
Pavoni, Vittorio, et al. "Outcome predictors and quality of life of severe burn patients admitted to intensive care unit." Scandinavian journal of trauma, resuscitation and emergency medicine 18.1 (2010): 1.
Surovtsev, V. I., et al. "Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data."Biochemistry (Moscow) 72.9 (2007): 989-993
</div> </body>
Lysostaphin
Lysostaphin is a 27 kDa-zinc metalloproteinase (EC 3.4.24.75) produced by Staphylococcus simulans. This enzyme hydrolyses glycine/glycine bonds in the pentaglycine interpeptide that maintain the stability of peptidoglycans in staphylococcal cell wall (Surovtsev 1).
Usage
In spite of recent advances in medical care, data reveals that mortality rate among burned patients remains higher depending on the extent of affected area (Pavoni 2). These death rates could also be driven by prolonged antibiotic courses, which related to immunocompetence decrease. Consequently, leading patient to a vulnerability state of the skin to microorganisms (Baker 3), besides the per se lost of the first line of antimicrobial defense. Given the fact that hospital acquired infections is a growing concern in public health alongside the development of resistant strains, burned individuals are at the risk top risk among patients (Leseva 4).
Biology
Lysostaphin is encoded by a three-modular gene that encompasses a self-cleavage peptide, a propetide and the self-coding sequence of lysostaphin. After protein processing, mature lysostaphin (246 aminoacids) is released and exhibits a triple-enzymatic activity: glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine. Consequently, it rapidly lyses actively growing and non-dividing cells including staphylococci in biofilms and, due to its specificity, it could have high potential in the treatment of antibiotic-resistant staphylococcal infections (Kumar 5).
Perspectives
This protein with antibiotic properties seems as a promissory candidate for medical purposes that combined with novel chimeric spider silk as polymeric matrix could become an interesting approach to address nosocomial infections by resistant strains.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 505
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
It’s important to take into account that this sequence encompasses only mature lysostaphin.
As our project relies on the advantage of Chlamydomonas reinhardtii as a proper chassis for high GC-content genes, our team adapted mature coding sequence of lysostaphin and codon optimized for microalgae. Notice that stop codon was removed from original sequence.
Cloning into pSB1C3 vector
For further details, explore our complete experimental part!
Analytical digestion
After several trials of transformation in DH-5α cells, plasmid bearing lysostaphin sequence was obtained. Analytical digestion was normally performed with about 500ng of plasmid.
Figure 1. Analytical digestion pSB1C3 + Lysostaphin
PCR confirmation
From plasmid backbone, PCR reaction was performed in order to identify the insert. Either our home-made X7 and Q5 polymerase worked perfectly.
Figure 2.PCR from pSB1C3 backbone
Sequencing
Through the last months, lysostaphin feed our hopes and became one of our favorite parts. In order to confirm that BBa_K2136002 was cloned properly, sequencing was performed under standard protocol provided by local service.
Sequencing with forward primer
Figure 3.Sequencing reaction for forward primer
Sequencing with reverse primer
Figure 4.Sequencing reaction for reverse primer
For further details of sequencing data, check here!
References
Baker, Christopher C., Carol L. Miller, and DONALD D. TRUNKEY. "Predicting fatal sepsis in burn patients." Journal of Trauma and Acute Care Surgery 19.9 (1979): 641-648.
Hojckova, Katarina, Matej Stano, and Lubos Klucar. "phiBIOTICS: catalogue of therapeutic enzybiotics, relevant research studies and practical applications." BMC microbiology 13.1 (2013): 1.
Leseva, M., et al. "Nosocomial infections in burn patients: etiology, antimicrobial resistance, means to control." Ann Burns Fire Disasters 26.1 (2013): 5-11.
Kumar, Jaspal K. "Lysostaphin: an antistaphylococcal agent." Applied microbiology and biotechnology 80.4 (2008): 555-561.
Hardy J, Römer L, Scheibel T (2008) Polymeric materials based on silk proteins. Polymer 49 (20): 4309-4327. DOI: 10.1016/j.polymer.2008.08.006
Kluge J, Rabotyagova O, Leisk G, Kaplan D (2008) Spider silks and their applications. Trends in Biotechnology 26 (5): 244-251. DOI: 10.1016/j.tibtech.2008.02.006
Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g
Pavoni, Vittorio, et al. "Outcome predictors and quality of life of severe burn patients admitted to intensive care unit." Scandinavian journal of trauma, resuscitation and emergency medicine 18.1 (2010): 1.
Surovtsev, V. I., et al. "Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data."Biochemistry (Moscow) 72.9 (2007): 989-993
</div> </body>
None |