Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli

Balaban, Cecilia Lucía; Suárez, Cristian Alejandro; Boncompain, Carina Andrea; Peressutti-Bacci, Natalia; Ceccarelli, Eduardo A.; Morbidoni, Héctor Ricardo

doi:10.1186/s12934-022-01766-9

Cited by 10 publications

(8 citation statements)

References 53 publications

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“…In this context, N-L is an anionic surfactant with multiple applications in biotechnology and biomedicine, including transdermal drug delivery [27][28][29], fabrication of biomaterials and nanomaterials [30][31][32], structural protein analysis [33,34], separation of co-purified tag-less proteins from protein complexes [35]and mild protein solubilization/extraction [36][37][38], among many others. In fact, it was the first non-denaturing agent used in the solubilization of recombinant proteins from IBs [39].…”

Section: Discussionmentioning

confidence: 99%

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography

et al. 2023

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Background Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products. Results Aiming at improving the purification of such difficult proteins, a novel procedure has been developed based on supplementing crude cell extracts with non-denaturing concentrations of the anionic detergent N-Lauroylsarcosine. The incorporation of this simple step in the downstream pipeline results in a substantial improvement of the protein capture by affinity chromatography, an increase of protein purity and an enhancement of the overall process yield, being the detergent not detectable in the final product. Conclusion By taking this approach, which represents a smart repurposing of N-Lauroylsarcosine applied to protein downstream, the biological activity of the protein is not affected. Being technologically simple, the N-Lauroylsarcosine-assisted protein purification might represent a critical improvement in recombinant protein production with wide applicability, thus smothering the incorporation of promising proteins into the protein market.

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Section: Discussionmentioning

confidence: 99%

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography

et al. 2023

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“…Studies have been conducted to improve the existing E. coli-based protein expression. Balaban et al demonstrated the use of osmolytes, such as N-lauroylsarcosine, to increase the expression rate of E. coli C43 (DE3) from the existing 30-40% to about 54% (Balaban et al, 2022). Engineered strains of E. coli BL21 (DE3), viz., BL21Star (DE3) that offers enhanced mRNA stability, BL21trxB (DE3) that promotes correct folding of proteins and formation of disulphide bonds, and BL21 (DE3) pLysS that lowers the leaky expression of the gene of interest, are examples of E.coli expression systems that have been developed and found superior to their counterparts (Jia and Jeon, 2016).…”

Section: Expression Systems For the Production Of Recombinant Lysinsmentioning

confidence: 99%

Section: Expression Systems For the Production Of Recombinant Lysinsmentioning

confidence: 99%

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Beyond antibiotics: phage-encoded lysins against Gram-negative pathogens

Shah,

Das,

Chavan

et al. 2023

Front. Microbiol.

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Antibiotics remain the frontline agents for treating deadly bacterial pathogens. However, the indiscriminate use of these valuable agents has led to an alarming rise in AMR. The antibiotic pipeline is insufficient to tackle the AMR threat, especially with respect to the WHO critical category of priority Gram-negative pathogens, which have become a serious problem as nosocomial and community infections and pose a threat globally. The AMR pandemic requires solutions that provide novel antibacterial agents that are not only effective but against which bacteria are less likely to gain resistance. In this regard, natural or engineered phage-encoded lysins (enzybiotics) armed with numerous features represent an attractive alternative to the currently available antibiotics. Several lysins have exhibited promising efficacy and safety against Gram-positive pathogens, with some in late stages of clinical development and some commercially available. However, in the case of Gram-negative bacteria, the outer membrane acts as a formidable barrier; hence, lysins are often used in combination with OMPs or engineered to overcome the outer membrane barrier. In this review, we have briefly explained AMR and the initiatives taken by different organizations globally to tackle the AMR threat at different levels. We bring forth the promising potential and challenges of lysins, focusing on the WHO critical category of priority Gram-negative bacteria and lysins under investigation for these pathogens, along with the challenges associated with developing them as therapeutics within the existing regulatory framework.

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“…These characteristics, in particular, make them promising alternatives to antibiotics [9,10]. In this context, the expression of endolysin from different phages via microbial cells as a chassis has attracted the attention of an increasing number of researchers [11][12][13][14]. A number of Salmonella-specific endolysins have been identified and characterized recently (Additional file 1: Table S1).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Salmonella endolysin XFII produced by recombinant Escherichia coli and its application combined with chitosan in lysing Gram-negative bacteria

et al. 2022

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Background Salmonella is a common foodborne pathogen, which can cause intestinal diseases. In the last decades, the overuse of antibiotics has led to a pandemic of drug-resistant bacterial infections. To tackle the burden of antimicrobial resistant pathogens, it is necessary to develop new antimicrobial drugs with novel modes of action. However, the research and development of antibiotics has encountered bottlenecks, scientific hurdles in the development process, as well as safety and cost challenges. Phages and phage endolysins are promising antibacterial agents that can be used as an alternative to antibiotics. In this context, the expression of endolysin derived from different phages through microbial cells as a chassis seems to be an attractive strategy. Results In this study, a new endolysin from the Salmonella phage XFII-1, named XFII, was screened and obtained. The endolysin yield exceeded 100 mg/mL by heterologous expression from E. coli BL21 and short induction. The endolysin XFII exhibited high bactericidal activity at a concentration of 0.5 μg/mL and reduced the OD600 nm of EDTA-pretreated E. coli JM109 from 0.8 to 0.2 within 5 min. XFII exhibited good thermo-resistance, as it was very stable at different temperatures from 20 to 80℃. Its bactericidal activity could keep constant at 4 °C for 175 days. In addition, the endolysin was able to exert lytic activity in eutrophic conditions, including LB medium and rabbit serum, and the lytic activity was even increased by 13.8% in 10% serum matrices. XFII also showed bactericidal activity against many Gram-negative bacteria, including Salmonella, E. coli, Acinetobacter baumannii, and Klebsiella pneumoniae. Surprisingly, the combination of endolysin XFII and chitosan showed a strong synergy in lysing E. coli and Salmonella without EDTA-pretreatment, and the OD600 nm of E. coli decreased from 0.88 to 0.58 within 10 min. Conclusions The novel globular endolysin XFII was screened and successfully expressed in E. coli BL21. Endolysin XFII exhibits a broad lysis spectrum, a rapid and strong bactericidal activity, good stability at high temperatures and under eutrophic conditions. Combined with chitosan, XFII could spontaneously lyse Gram-negative bacteria without pretreatment. This work presented the first characterization of combining endolysin and chitosan in spontaneously lysing Gram-negative bacteria in vitro.

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Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli

Cited by 10 publications

References 53 publications

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography

Beyond antibiotics: phage-encoded lysins against Gram-negative pathogens

Characterization of Salmonella endolysin XFII produced by recombinant Escherichia coli and its application combined with chitosan in lysing Gram-negative bacteria

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