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Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3).

Kashani HH, Moniri R - Osong Public Health Res Perspect (2015)

Bottom Line: Cloning accuracy was confirmed by electrophoresis.Observation of a 28.5 kDa band confirmed LysK protein expression.In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

View Article: PubMed Central - PubMed

Affiliation: Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran.

ABSTRACT

Objectives: Bacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter.

Methods: The E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein.

Results: In this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression.

Conclusion: In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

No MeSH data available.


Related in: MedlinePlus

Analysis of LysK-CHAP protein expression with two methods. (A) LysK-CHAP protein expression was analyzed with SDS-PAGE. (B) LysK-CHAP protein expression was analyzed with Western blot. C = control; CHAP = cysteine/histidine-dependent amidohydrolase/peptidase; SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis; M = protein Marker; S = protein sample.
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fig2: Analysis of LysK-CHAP protein expression with two methods. (A) LysK-CHAP protein expression was analyzed with SDS-PAGE. (B) LysK-CHAP protein expression was analyzed with Western blot. C = control; CHAP = cysteine/histidine-dependent amidohydrolase/peptidase; SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis; M = protein Marker; S = protein sample.

Mentions: Observation of a 28.5 kDa protein band confirmed LysK-CHAP protein expression (Figure 2).


Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3).

Kashani HH, Moniri R - Osong Public Health Res Perspect (2015)

Analysis of LysK-CHAP protein expression with two methods. (A) LysK-CHAP protein expression was analyzed with SDS-PAGE. (B) LysK-CHAP protein expression was analyzed with Western blot. C = control; CHAP = cysteine/histidine-dependent amidohydrolase/peptidase; SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis; M = protein Marker; S = protein sample.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588433&req=5

fig2: Analysis of LysK-CHAP protein expression with two methods. (A) LysK-CHAP protein expression was analyzed with SDS-PAGE. (B) LysK-CHAP protein expression was analyzed with Western blot. C = control; CHAP = cysteine/histidine-dependent amidohydrolase/peptidase; SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis; M = protein Marker; S = protein sample.
Mentions: Observation of a 28.5 kDa protein band confirmed LysK-CHAP protein expression (Figure 2).

Bottom Line: Cloning accuracy was confirmed by electrophoresis.Observation of a 28.5 kDa band confirmed LysK protein expression.In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

View Article: PubMed Central - PubMed

Affiliation: Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran.

ABSTRACT

Objectives: Bacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter.

Methods: The E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein.

Results: In this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression.

Conclusion: In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

No MeSH data available.


Related in: MedlinePlus