|Year : 2015 | Volume
| Issue : 2 | Page : 72-77
Cytoplasmic expression, optimization and catalytic activity evaluation of recombinant mature lysostaphin as an anti-staphylococcal therapeutic in escherichia coli
Samaneh Naderi1, Mohammad Yousef Alikhani2, Jamshid Karimi3, Nooshin Shabab4, Nejad Mohamadi1, Hossein Zarei Jaliani5, Massoud Saidijam6
1 MSc Student of Medical Biotechnology, Department of Molecular Medicine and Research Center for Molecular Medicine, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
2 Associate Professor, Department of Microbiology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
3 Assistant Professor, Department of Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
4 BSc in Biology, Department of Molecular Medicine and Research Center for Molecular Medicine, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
5 Assistant Professor, Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
6 Department of Molecular Medicine and Research Center for Molecular Medicine, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
|Date of Web Publication||5-Jul-2017|
Department of Molecular Medicine and Research Center for Molecular Medicine, School of Medicine, Hamadan University of Medical Sciences, Hamadan
Source of Support: None, Conflict of Interest: None
Objective: Scientists are to develop novel antibacterial therapeutics in order to eliminate medically significant pathogens resistant to antibiotic. Lysostaphina zinc metalloprotease with the sub-atomic weight of 27kDa and particularly tic action against Staphylococcus aureus degrades the S. aureus by hydrolyzing the pentaglycine cross-links introduce in its cell wall. Because of such potential, lysostaphin will be a decent agent for treatment of antibiotic-resistant staphylococcal infections. Also, due to the broad needs of society to increase localization of sciences, the national production of this drug in research laboratories would be necessary. Materials and Methods: First, with the aid of articles we identified Lysostaphin sequences without signal peptide via Gene Bank data base. In order to achieve the correct protein sequence, we add the entrokinase cutting site to our sequence. To ensure the correct cloning process, the entire process of cloning and protein expression was evaluated in silico. The sequence obtained for the synthesis was sent to the Biomatik Company in Canada (BIOMATIK). After the primer design and synthesis of the DNA fragment of the gene, we amplified the gene using PCR. The mature lysostaphin gene was cloned in plasmidpET28a and expressed in E.coli with the carboxyl terminal hexa-histidine fusion tag under the transcriptional control of T7/lac promoter/operator. Result: The transformed E.coli BL2 (DE3) cells produced catalytically active recombinant lyso staphin after being induced by IPTG. Conclusion: This study shows that the E. coli expression system is suitable for expression of recombinantly so staphin and according to the conducted bioassay in this study, the expressed protein can be considered as an effective therapeutic agent against antibiotic resistant staphylococcus aureus.
Keywords: Antibiotic resistance, Genetic Engineering, MRSA, Recombinant Protein, Therapeutics
|How to cite this article:|
Naderi S, Alikhani MY, Karimi J, Shabab N, Mohamadi N, Jaliani HZ, Saidijam M. Cytoplasmic expression, optimization and catalytic activity evaluation of recombinant mature lysostaphin as an anti-staphylococcal therapeutic in escherichia coli. Acta Med Int 2015;2:72-7
|How to cite this URL:|
Naderi S, Alikhani MY, Karimi J, Shabab N, Mohamadi N, Jaliani HZ, Saidijam M. Cytoplasmic expression, optimization and catalytic activity evaluation of recombinant mature lysostaphin as an anti-staphylococcal therapeutic in escherichia coli. Acta Med Int [serial online] 2015 [cited 2019 Jul 16];2:72-7. Available from: http://www.actamedicainternational.com/text.asp?2015/2/2/72/209656
| Introduction|| |
Staphylococcus aureus is the major reason for nosocomial and community acquired infections and a number of other diseases.,,, It is been found that methicillin-resistant Staphylococcus aureus (MRSA), even shows strength to the latest staphylococcal treatment strategy; vancomycin. Like wise, antibiotic resistance among other strains of medically important pathogens has become a global dilemma therefore scientists are to develop new antibacterial agents to eliminate such drug-resistant microorganisms. A very interesting category of anti bacterials is enzybiotics with their novel mode of antibacterial action and the capacity to kill resistant bacteria. There are major types of enzybiotics, among them; bacteriocins are the main focus of this study. Bacteriocins are proteins produced by one strain of bacteria to prevent the growth of other bacterial strains. The special bacteriocin whose expression and activity is studied in this paper, is Lysostaphin; a single polypeptide with glycyl glycine endopeptidase activity that has been identified and isolated from Staphylococcuss imulans biovar staphylolyticus [NRRLB-2628(gi|126496)] for the first time. Plasmid p ACK1 carries lysostaphin gene and when expressed, it produce sapre-proenzyme with a 36-residue signal peptide and 15 tan dem repeats that result in the mature form of lysostaphin with 247aminoacids by their removal. Recombinant production of lysostaphin with the aim of further reassessments of its clinical potentials is very important and recombinant DNA technology not only helps to produce abundant quantities of therapeutic proteins but also provides the possibility of process ingand engineering of produced proteins. The present study was performed to localize the production of recombinant lysostaphin through recombinant DNA technology followed by an initial evaluation of its bactericidal effect in order to under lie further investigation so fits therapeutic application against antibiotic resistant bacterial strains.
| Materials and Methods|| |
The syntheticly so staphin gene (Sae) (Biomatik, Canada) including restriction enzyme recognition sites and His-tag fusion partner was constructed with the sequence similar to the wild-type gene sequence (U66883.1) encoded in Staphylococcus imulans biovar staphylolyticus bacterials train and the Lysc DNA was PCR amplified (BIORAD Thermal cycler, USA) using taq DNA polymerase and designed primers LsFP5'-GGC TCA CCA TGG ATG GCT GCA ACA CAT GAA CAT TC-3'and LsRP5'- GGC TCG AAG CTT CTT TAT AGT TCC CCA AAG AAC-3'.The synthetic Lysc DNA was used as a template. The amplification conditions were 35 cycles of 94°C for 45s, 58.6°C for 45s, and 72°C for 60s and the final extension was kept at 72°C for 7 min. The PCR product visualized by gel electrophoresis on 1% agarose gel.
Cloning of Lysgeneinp ET28 a Vector
PCR product was purified from agarose gel by the agarose gel purification kit (Bioneer, Korea) and digested by NcoI (Ferment as, Germany) and Hind III (Ferment as, Germany) and pET28a(+) expression vectoral so was similarly digested and purified. The concentration of both PCR product and plasmid were assessed by Nanodrop (Bio Tek, USA). The purified DNA fragment was clone din to the pET28a(+) expression vector by T4DNA ligase (Ferment as, Germany) and ligated products were transformed into the E. coli Top 10F'. Screening was performed by colony PCR. Recombinant plasmids were confirmed by restriction digestion and sequencing (Biomatik, Canada). Sequencing data is available.
Expression was analyzed in total cell extract at both RNA and protein level. For this purpose, E. coli BL21(DE3) cells were transformed with recombinant vectors by heat shock and were grown at 220 rpm and 37°C in 50 ml LB broth medium containing 50 μg/ml kanamycin (10 mg/ml final concentration) for 12-16 h. Then IPTG (1 m M final concentration) was added to the bacterial culture Satan optical density of 0.4-0.6. to induce the expression of recombinant protein and incubated for 4 h at 37°C and agitation speed of 220 rpm. Cellular pellets were collected by centrifugation at 4°C 9000 rpm for 5 min 2 h and 4 h post induction. First, total RNA was extracted by commercial RNX-Plus and c DNA was synthesized by Revert Aid M-Mul Reverse Transcriptase and Random Hexamer Primers according to our lab protocol, PCR was performed by use of c DNA as template and the product of PCR visualized on 1% agarose gel. Expression at protein level was analyzed by SDS-PAGE, so that samples were prepared with SDS Loading buffer 1X and the same amount of each sample (20 μl) was loaded on 15% sodium dodecyl sulfate polyacrylamide gel. After electrophoresis, gel was stained with Co omassie brilliant blue.
Optimization of Recombinant Protein Expression
Optimization of recombinant protein expression was performed by changing various parameters such as harvesting time following IPTG induction and concentration of IPTG to find the optimal conditions for the expression of recombinant protein. Different IPTG concentrations (0.1-1 mM) and harvesting times after induction (4, 8, 12 and 16 hours) were examined.
Immuno-blotting of Recombinant Protein
Total cell extract of 2 ml bacteria was prepared with SDS loading buffer and loaded on 15% sodium dodecyl sulfate polyacrylamide gel and transferred to an its cellulose membrane in 4 h using transfer buffer and electrophoresis. Then nitro cellulose membrane was blocked with 5% non fat milk at room temperature for 2 h. Membrane was washed 3 times with PBS-T wean 20 and then incubated for 2 h with diluted (1:1000) HPR- labeled monoclonal anti His-tag antibody (R&D, USA) at room temperature (22°C) and was washed 3 times. Final reaction detection was assessed by using diamino benzidine tetra hidro chloride (DAB; Sigma, USA) substrate which yields an insoluble brown product.
Lyso Staphin Enzymatic Biobassay
In order to assess the antibacterial effects of staphinonlysis of resistant S. aureus, MRSA colonies were grown on blood agar for 18-24 h at 37°C and were randomly selected and transferred to physiological serum (McFarland standard 0.5). Sterile swab was dipped in an S.aureus suspension (McFarland standard 0.5) and plated on to Muller Hinton agar. Antibiotic disks were saturated with 30 μl suspension of IPTG induced E. coli colonies, containing recombinant lyso staphin and placed on the plate 15 mm far away from each other. The lysis pattern of recombinantly staphin was observed after 16-18 h incubation at 37°C.
| Results|| |
Gene Amplification and Cloning of lys Gene in pET28a Vector
The gene encoding mature lyso staphin was PCR amplified using designed primers and 800 bp PCR product visualized on a 1% agarose gel [Figure 1].
|Figure 1: Agarose gel electrophoresis of PCR product on 1% agarose gel, l an esA, PCR product of amplified Lys gene (800bp); B, negative control; C, 100bp DNA molecular size marker (Ferment as, Lithuania)|
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Recombinant plasmids were extracted from positive colonies and digested with NcoI and Hind III restrict ion enzymes, and the presence of 800 bp fragment confirmed the cloning of Lysgene into the vector pET 28 a (+) [Figure 2].
|Figure 2: Double digestion of recombinant vector pET28a (+) with NcoI and Hind III restriction enzymes. Lanes A, 1 kb DNA molecular size marker (Ferment as, Germany); B, digested recombinant plasmid (plasmid at the top and gene at the bottom of gel); C, 100 bp DNA molecular size marker (Ferment as, Germany)|
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Recombinant Protein Expression and Optimization of Expression
Recombinant vectors pET28a-Lys were transformed into E. coli BL21(DE3) and IPTG induced expression of desired protein at different concentrations of IPTG and different harvesting post induction times was investigated at RNA and protein levels by RT-PCR and SDS-PAGE, respectively. The result of RT-PCR was showing in [Figure 3].
|Figure 3: RT-PCR, Lanes A, 100bp DNA molecular marker (Ferment as, Lithuania); B, Transformed E. coli BL21 before induction(negative control); C, Transformed E. coli BL21 after induction|
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The results of SDS-PAGE in [Figure 4] showed that the recombinant protein expression reached its highest level at the concentration of 0.5 and 1m MIPTG 4 h after induction (lanes 6 and 9) and the band in the region of 27 k Da of molecular weight marker was identified.
|Figure 4: Analysis of lysostaphin expression at different harvesting times and different concentration of IPTG. Lanes1, protein molecular size marker (Sigma- Aldrich, Color Burst™); 2, negative control (un induced BL21); 3,4,5,8, recombinant E. coli BL21 2 h after induction with IPTG0. 1, 0.3, 0. 5 and 1 mM; 6,9, recombinant E. coli BL214 h after induction with IPTG0.5 and 1 mM; 7 and 10, recombinant E. coli BL2116 h after induction with IPTG 0.5 and 1 mM|
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Immuno-blotting of Recombinant Protein
Immuno blotting with HPR-labeled monoclonal antiHis-tag antibody confirmed the identity of expressed recombinant protein at 27k Daregion [Figure 5].
|Figure 5: Western blotting analysis of expressed recombinant lysostaphin using antiHis-tagantibody. Lanes1, protein molecular size marker (Sigma Aldrich, ColorBurst™); 2, W Bnegative control (uninduced recombinant E. coli BL21); 3 and 4, recombinant E. coli BL21 induced by 4 h after induction with 0.5 m MIPTG; 5 and 6, E. coli BL21 cell lysate transformed by recombinant plasmid 4 h after induction with1m MIPTG|
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Lysostaphin Enzymatic Bioassay
In order to evaluate the lysis effect of expressed recombinant lysostaphin on methicillin resistant S.aureus (MRSA) growing and culture procedures were done as mentioned before and the following results were observed. Clear zones around disks enclosed with black circles, are due to the growth inhibition of MRSA caused by recombinant lysostaphin. As shown on the plate different concentration of IPTG and different harvesting time after induction has been observed in the conducted bioassay. The control disk contains un induced recombinant E.coli BL21. There sults are in accordance with those of the immune blotting and SDS-PAGE, recombinant lysotaphin shows its highest antibacterial activity at the concentration of 1m MIPTG and 4 h after induction [Figure 6].
|Figure 6: Lysis of S. aureus by E. coli BL 21 cell lysate above containing expressed recombinant lysostaphin. Numbers the fraction line show time after induction based on hour(h) and numbers under the line show the concentration of IPTG(mM) used for induction|
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| Discussion|| |
Lysostaphin (EC.188.8.131.52), an enzyme with the bactericidal activity against Staphylococcus aureus and other staphylococcal species, expressed in E.coli. In this study we selectively amplified the mature lysostaphin gene and clone din pET-28a(+) between NcoI and HindIII restriction sites. Various restriction enzymes and expression vectors were used in previous reports. For example, lysostaphin was expressed using the expression vector pET-15 band the NdeI and Bam HIsites or in another study that was conducted in 2009 in England, expression vector pET-21a and NdeI and XhoI sites were used to produce recombinantly so staphin there is no significant differences between different studies. The first step in the expression of every recombinant protein in vitro is to access the gene encoding that protein. In the present study, in order to clone the lysostaphin gene, a synthetic form of the gene was provided. In early studies, lysostaphin production methods were based on the purification of the extracts of S. simulans, which was likely to be infected with various pathogens and allergens. The production of lysostaphin precursor; proly so staphin as recombinant in B. subtilis and B. sphaericus has been reported previously. E. coli is considered to be a reliable expression host for the production of recombinant proteins, sow picked E. coli BL21(DE-3) in this study. Since lysostaphin has a bacterial origin it doesn't require certain engineering or post translational modification, therefore E. coli system considered to be suitable for expression of lysostaphin. It is proposed that pET expression system is a proper choice for the expression of the protein. Various reports have been presented the expression of lysostaphinin E. coli using lysostaphin promoter., CMV promoter has also been reported for lysostaphin expression, in another report. In another study, lysostaphin was expressed in mice and secreted into milk after special manipulations. In the present study we selectively amplified the mature lysostaphin gene and expressed it under transcriptional control of T7/Lac promoter/operator., The ability to quickly remove S. aureus nasal colonization has a significant impact on the prevention of life-threatening infections and the spread of drug-resistant Staphylococcus aureus in hospitals and community. Effective assessment of staphylococcal treatment requires appropriate animal models. The use of animal models and evaluation of recombinant lysostaphin in vivo condition is very important. In order to further study the efficacy of recombinant lysostaphin, purification is required following approval of expression. Several purification procedures of lysostaphin from S. simulans cell culture have been reported so far.
In this study, His6-tag fusion was used with the aim to facilitate the future procedure of purification of the recombinant protein. Lysostaphin was over expressed and purified in a study conducted in 2001 by Szweda and colleagues. Different types of proteins have been used as fusions in these studies.
We also investigated the anti-staphylococcal activity of lysostaphin by disk diffusion. Besides its anti-staphylococcal activity, lysostaphin has been commonly used for rapid screening of S. aureus from other staphylococcal species in clinical labs.,,,, Recently at wo-step labeling protocol using lysostaphin and bioorthogonal click chemistry for staining bacteria has been applied successfully. Because of specificity of lysostaphin-N3, it binds efficiently to Staphylococcus aureus and in the next step Azido (N3) will click to DIBO thus S. aureus will selectively labeled. It can also be utilized in food industry as apreservative. According to the previous studies on the potential clinical applications of lysostaphin as an ovel effective therapeutic.,,,, we suggest future studies on figuring out the high efficient and low costs methods for producing large amounts of pure preparations of lysostaphin as well as looking for yielding purification methods and further studies are required to explore the proper formulations of lysostaphin as a therapeutic drug indifferent forms such as topical ointments or oral administrations., Crystallized Structure of this protein has been confirmed its function as an antibacterial agent. Based on the results of this research, enzyme engineering could be applied to increase its antibacterial activities and also to improve its stability in solutions. More investigations have been concentrated on eradication of S. aureus biofilms using lysostaphin enzyme. The motile bacteria such as Bacillus Thu ringiensis which expressedly so staphin, could be applied with drugs that may usually have difficulty in penetrating the biofilm, to facilitate penetration of lysostaphinin to the inner parts of the biofilm. So the next step of this study could be to purify this enzyme and also to try to transfer prepared construct to other hosts specially motile bacteria.
| Conclusion|| |
Following the significant growth of bacterial strains resistant to chemical antibiotics, there is an urgent need to find alternative low cost solutions with high performance and availability for production of useful antibiotics using genetic engineering techniques and further efforts to achieve the appropriate ways for purification and commercial production of these agents and replacement of these products with conventional treatments are crucial. Moreover, Protein stability and half-life are essential factors in the application of a protein as a therapeutic, therefore, it is necessary to estimate the stability of lysostaphin at different conditions of pHand temperature also more understanding of the functional and structural features of lysostaphin will be helpful in the standardization of its appropriate formulations to be used against antibiotic-resistant S. aureus strains in future studies.
| Acknowledgements|| |
The present study was financially supported by the deputy of research and technology, Hamadan University of Medical Sciences. The results described in this paper were part of student thesis.
| Abbreviations|| |
MRSA, methicillin-resistant Staphylococcus aureus; DAB, 3,3'-diaminobenzidine; HRP, horse radish peroxidase; IPTG, isopropyl-β-thiogalactopyranoside; LB, Luria-Bertanibroth.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]