Production of Lysostaphin by Nonproprietary Method Utilizing a Promoter from Toxin–Antitoxin System
Lysostaphin is a staphylolytic protein of growing interest from biotechnological and pharmaceutical industry due to its potential use in preventing and combating staphylococcal infections. Here, we describe an optimized method for production of lysostaphin in an inductionless system utilizing constitutive promoter from staphylococcal toxin–antitoxin system PemIK-Sa1. We investigated the influence of ribosome-binding site sequence, Escherichia coli producer strain and growth media on yield and kinetics of recombinant protein production. Lysostaphin was purified in its native active form using one-step cation-exchange chromatography. The system provides a method for cost-efficient and scalable protein production, and can be applied to produce other biotechnologically significant proteins.
KeywordsLysostaphin Protein expression Ion exchange chromatography Staphylococcus Recombinant protein Toxin–antitoxin system
Many different expression systems have been developed to date, but the production of recombinant proteins in Escherichia coli remains the most widely used . E. coli offer high growth rate and inexpensive substrates which allow for scalable and cost-efficient production. Additionally, broad prevalence of the system results in availability of numerous host strains and variety of expression vectors.
Most expression systems rely on inducible promoters enabling control over the onset of protein production. Biomass is usually first produced and only then the culture is switched to production. Apart from clear advantages, the inducible systems, including the most widely utilized lac promoter , its derivatives, tac/trc promoter [3, 4] and ara promoter , require expensive inducers, a considerable burden in large scale production. Other inducible promoters respond to temperature , pH  or depletion of particular substrates [8, 9], but provide less stringent control and have not been widely used.
Tightly controlled strong promoters have been conventionally utilized , although often result in inactive, insoluble protein, deposited in inclusion bodies [11, 12]. Additionally, high level of mRNA can lead to ribosome destruction  and metabolic burden associated with overexpression  may lead to cell death. An alternative approach uses constitutive promoters. Continuous production from weak promoters allows gradual accumulation and often more efficient folding. Another advantage is in cost reduction by eliminating the inducers.
Lysostaphin is a bacteriolytic metalloprotease originating from Staphylococcus simulans biovar staphylolyticus. It degrades the cell wall of multiple species of staphylococci by hydrolysis of pentaglycine crosslinks within peptidoglycan [15, 16]. The gene encoding lysostaphin (end) is located on pACK1 plasmid together with epr gene which provides host resistance. The resistance is ensured by increased number of serine residues in the cross bridges [17, 18]. Native lysostaphin is composed of mature polypeptide of 246 amino acid residues, propeptide of 211 residues and signal peptide of 36 residues . The signal peptide is removed upon secretion and the propeptide is processed outside the cell by an extracellular cysteine protease [20, 21]. The mature lysostaphin is a protein of 27 kDa and isoelectric point of approximately 9.5 .
Lysostaphin had been shown effective in treatment of staphylococcal infections [23, 24] and reduction of staphylococcal carriage [25, 26], but has not been translated to clinics. After a period of relative stagnation, medical lysostaphin is being rediscovered against multidrug-resistant MRSA (Meticillin-resistant Staphylococcus aureus) and VRSA (Vancomycin-resistant Staphylococcus aureus) [27, 28], as well as notoriously recalcitrant biofilms [29, 30]. Latest concepts incorporate lysostaphin into medically relevant materials, which include coating of orthopedic implants with polymer matrix containing lysostaphin and manufacturing gels or wound-dressing materials for the treatment of topical or wound infections [31, 32, 33, 34, 35]. Lysostaphin-containing materials were proven useful in both preventing and eradicating S. aureus infections linked to orthopedic implants in murine model. Furthermore, a biopolymer impregnated with lysostaphin had the antistaphylococcal activity comparable to commercially available antimicrobial wound dressings . The growing interest in lysostaphin as an antistaphylococcal agent calls for a cost-effective and scalable method of production.
Aside potential clinical use, lysostaphin is an indispensable tool in research and diagnostics. Lysostaphin treatment remains the most effective method for lysing staphylococcal cells. This is because other common methods including alkaline lysis, sonication and homogenization are ineffective for staphylococci . Lysostaphin is thus essential for extraction of nucleic acids and intracellular proteins. The enzyme is currently available from commercial sources, although relatively expensive. As a result, laboratories with sufficient experience may benefit from a non-complicated and reliable expression system to produce the enzyme on their own.
Several methods of lysostaphin expression and purification have been described to date. The enzyme was purified from Staphylococcus simulans conditioned media [38, 39, 40, 41, 42], however, with poor yield. Additionally, such produced enzyme may contain contaminating allergens and pyrogens. Alternatively, production in heterologous hosts has been reported including E. coli [43, 44, 45, 46, 47, 48, 49, 50], Bacillus subtilis and Lactobacillus casei , Pichia pastoris  and even in mammalian cells . The most promising system in terms of efficient large scale production was developed in E. coli and based on an inducible promoter. Prior proposed systems required multi-step purification and often relied on immobilized metal ions chromatography, which is known to compromise lysostaphin activity due to leakage of metal ions, which are lysostaphin inhibitors . No system based on a constitutive promoter and providing single-step purification has been proposed to date.
Toxin–antitoxin (TA) systems are widely spread among bacteria and contribute to maintenance of mobile genetic elements, mediate phage infection defense, stress adaptation  and other functions. While investigating the staphylococcal pemIK-Sa1 toxin–antitoxin system  we observed continuous expression of GFP (green fluorescent protein) under pemIK-Sa1 promoter increasing with culture density. This attractive characteristics prompted us to test the utility of the promoter in circumventing the need of inducible expression for efficient recombinant protein production.
In this study, we provide an efficient expression system for recombinant lysostaphin in E. coli. The system utilizes a constitutive, non-inducible promoter from pemIK-Sa1 toxin–antitoxin system. Furthermore, the system is constructed using an easily available pUC18 backbone suitable for cost-effective commercial production of recombinant proteins. Furthermore, we introduce a one-step ion exchange chromatography purification resulting in highly active and pure preparation. Altogether, our system allows rapid, efficient, scalable and cost-effective production of recombinant lysostaphin, which may easily be adapted for use in pharmaceutical and biotechnological applications.
Materials and Methods
Bacterial Strains and Culture Conditions
E. coli Top10 and DH5α were used for cloning and plasmid propagation, respectively. Both above-mentioned strains and BL21(DE3) were used to test protein expression. The bacteria were cultivated in Luria–Bertani Broth (LB, Sigma) or Tryptic Soy Broth (TSB, Sigma). The media were supplemented with 100 μg/ml ampicillin to assure plasmid maintenance. The cultures were cultivated at 37 °C with shaking.
Construction of Expression Vectors
Sequence of primers used in this study
GFP and the mature lysostaphin coding sequences were cloned into pUC18 vector. Additionally, lysostaphin with C-terminal His-tag was prepared. The sequences were amplified with following pairs of primers: GFPSacI and GFPPstI for GFP, and lizoSacI_For and lizoPstI_Rev or lizoSacI_For and lizoHisPstI_Rev for lysostaphin. Plasmids pALCP2G  and pBADLys  encoding GFP and lysostaphin, respectively, were used as templates. The PCR products and pUC18 plasmid were digested with SacI and PstI (Thermo Scientific) and ligated with T4 DNA ligase (Thermo Scientific). Resulting constructs were denoted as pUC18/GFP, pUC18/Lysostaphin and pUC18/Lysostaphin-His6.
Regulatory sequences were transferred from pTZ57R-based plasmids prepared before into pUC18/GFP, pUC18/Lysostaphin and pUC18/Lysostaphin-His6. All plasmids were digested with EcoRI and SacI and the regulatory sequences were ligated upstream the protein coding sequence. This resulted in a construct for expression of GFP and four constructs for expression of lysostaphin, namely pUC18/promEco/GFP, pUC18/promEco/Lysostaphin, pUC18/promEco/Lysostaphin-His6, pUC18/promSau/Lysostaphin and pUC18/promSau/Lysostaphin-His6. Best constructs for recombinant lysostaphin production (pUC18/promEco/Lysostaphin and pUC18/promSau/Lysostaphin) were deposited in Addgene (IDs 125765 and 125766, respectively).
Lysostaphin Activity Assay
Lysostaphin activity was determined by monitoring the decrease in the optical density (OD) at 600 nm of suspension of susceptible S. aureus cells. An overnight culture of S. aureus RN4220 was diluted to an optical density of 1 in 0.1 M phosphate buffer, pH 7.5. The sample tested was diluted 10 times in L buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) in a 96-well plate. In each case 100 μl of the tested sample dilution was mixed with 100 μl of S. aureus cell suspension. The OD was followed with a microplate reader (PowerWaveXSelekt, Bio-Tek Instruments) for 30 min. The activity was expressed in arbitrary units calculated as follows. The blank-corrected Δ OD600nmt0 − t30 was set as a value of 100 and for each time point the respective Δ OD600nm the proportional value from the range 100 to 0 was assigned. Activity is expressed as 50 (corresponding to the half of initial OD600nm value) divided by the time (in minutes) when OD600nm reached the value.
GFP Reporter Assay for Determination of Promoter Activity
Overnight cultures of E. coli Top10, DH5α and BL21(DE3) cells transformed with pUC18/promEco/GFP were diluted 1:100 in 20 ml of LB supplemented with 100 μg/ml ampicillin and cultivated at 37 °C with shaking. Samples of 0.5 ml were collected each hour. The cells were pelleted, resuspended in 0.5 ml of phosphate-buffered saline (pH 7.4) and sonicated (30 pulses, 500 ms, 80% amplitude) using UP50H ultrasonic processor (Hielscher). The fluorescence of the lysates was determined with PowerWave microplate reader (Biotek) at 480 nm (excitation) and 510 nm (emission).
Optimization of Protein Production
To establish the best conditions for lysostaphin production, the kinetics of the recombinant protein expression from pUC18/promEco/Lysostaphin and pUC18/promEco/Lysostaphin-His6 plasmids was examined. Bacterial host strain, the growth medium and the time of cultivation were subject of optimization. Lysostaphin production was monitored by determining the staphylolytic activity of tenfold diluted lysate as well as by SDS-PAGE of the whole lysate, and its soluble and insoluble fractions.
The overnight cultures were diluted 1:100 in 50 ml of tested medium supplemented with 100 μg/ml ampicillin. The bacteria were grown at 37 °C with shaking and first sample (2 ml) was taken 4 h after inoculation. Next samples were taken every hour for subsequent 7 h. The last sample was taken 24 h since the start of the culture. Samples were pelleted by centrifugation and the pellets were suspended in 200 μl of buffer L. 50 μl of every sample was set aside for total cell protein analysis. The remaining 150 μl were sonicated (5 series of 30 pulses, 800 ms, 80% power) to obtain clear lysates. The samples were clarified by centrifugation (10 min, 4 °C, 15,000×g). The supernatant was used to assay the protein content of the soluble fraction while the pellet included the insoluble fraction. The pellets were resuspended in 100 μl of buffer L. All samples were analyzed by SDS-PAGE .
Production and Purification of Lysostaphin
Overnight culture of E. coli DH5α transformed with pUC18/promEco/Lysostaphin was diluted 1:100 in 200 ml of TSB supplemented with 100 μg/ml ampicillin and cultured at 37 °C with shaking for 16 h. The cells were pelleted, resuspended in 10 ml of buffer A (50 mM sodium phosphate, pH 7.5) and sonicated on ice (6 pulses of 30 s, 1 min intervals, maximum power) with UD-11 ultrasonic disintegrator (Techpan). The lysate was clarified by centrifugation (20 min, 4 °C, 15,000×g). The supernatant was applied (2 ml/min) onto 15 ml of cation-exchanger Source S30 resin (GE Healthcare Life Sciences) equilibrated with buffer A. The column was washed with 60 ml of buffer A. Elution was performed in linear gradient of buffer B (50 mM sodium phosphate, 1 M NaCl, pH 7.5) from 0 to 30% in a total volume of 150 ml. 2 ml fractions were collected. The fractions containing lysostaphin were pooled and concentrated 10 times using Amicon Ultra-15 Centrifugal Filter Units with MWCO of 3000 kDa (Merck). The preparation was dialyzed twice against buffer D (10 mM sodium acetate, pH 4.5) at 4 °C. The purified protein was aliquoted, frozen in liquid nitrogen and lyophilized (Christ LDC-1, Alpha 1–4 liophilizer). The protein concentration was determined by measurement of the absorbance at 280 nm (extinction coefficient: 2.43/cm/mg ml).
Staphylolytic activity of lysates from E. coli DH5α transformed with the plasmids encoding different variants of lysostaphin under the control of the pemIK-Sa1 promoter cultured in LB and TSB medium
Time post inoculation (h)
LB medium (A.U.)
TSB medium (A.U.)
In this study, we took advantage of the features of the promoter derived from staphylococcal PemIK-Sa1 toxin–antitoxin system to design an induction-free platform for efficient production of recombinant proteins. The utility of the platform was exemplified by expression of lysostaphin, an important antistaphylococcal agent attracting a growing interest of pharmaceutical and biotechnology industry [34, 35, 62].
RBS sequences allow to fine-tune protein expression. Often the expression of genes clustered in operons is varied by differing the RBS sequences. In TA systems for example, the antitoxin is usually produced in higher amounts than the downstream-encoded toxin . To bypass the expected low expression from non-canonical RBS, we optimized its original sequence to that characteristic for E. coli and S. aureus. Strikingly, we did not observe any difference in activity or kinetics of lysostaphin production from these two distinct RBS sequences when evaluated in E. coli. This indicates that in our particular example, the limiting feature is the strength of the promoter, but not the RBS.
The kinetics of protein production driven by the tested pemIK-Sa1 constitutive promoter matched that described previously for other staphylococcal toxin–antitoxin systems belonging to the same class, namely mazEF and savRS [64, 65]. The promoter was active during logarithmic growth phase. In the late stationary phase, degradation of recombinant protein was observed and for this reason the optimal time of harvest coincided with the peak of optical density at the early stationary phase.
Production of recombinant proteins is among biotechnological processes resulting in high value products. Factors influencing profitability include efficiency and the cost of an expression platform and downstream processing—such as purification . We provided the procedure based on generally accessible pUC18-based vectors and E. coli strains to produce mature lysostaphin in one-step purification. pUC18 is a high copy number plasmid which compensates the use of a relatively weak promoter.
A method of lysostaphin purification has been previously described based on immobilized metal ion affinity chromatography [46, 47, 48]. Histidine tagging has not affected the activity, but our results show that it may significantly affect protein stability. What is more, lysostaphin is a Zn2+-dependent metalloprotease and trace Ni2+ or Co2+ ions released from the resin during purification result in significant decrease in enzymatic activity by substituting the catalytic Zn2+ ion . Zn2+ affinity chromatography was proposed as an alternative [67, 68], but a two-step procedure including additional ion-exchange chromatography was necessary to obtain relevant purity. Not only the two-step procedure, but even the relatively high concentrations of imidazole necessary for elution substantially increase the costs of high-scale industrial purification. Moreover, it was recently reported that excess exogenous Zn2+ inhibits lysostaphin . Even the more, some applications require the recombinant protein in its native form (i.e. without expression and purification tags) while precise tag removal is often impossible or requires additional reagents (i.e. proteases) and steps (i.e. tag removal, protease removal) which substantially increase the production costs and contribute additional impurities to the final preparations . Here, we described a single-step, cation-exchange chromatography-based procedure yielding pure preparations of native sequence lysostaphin. Our approach overcomes the limitations of prior techniques proposed for lysostaphin purification. In the optimized conditions of pH 7.5, only a few proteins other than lysostaphin bind to the resin and none of those endogenous E. coli proteins co-eluted with the protein of interest. This phenomenon is explained by a bimodal distribution of isoelectric points (pI) in bacterial proteome . Nearly two-thirds of E. coli proteins have theoretical pI below 7.5 and thus exhibit negative or neutral charge at the pH used for purification excluding their binding to a cation exchanger. The majority of remaining E. coli proteins with higher theoretical isoelectric points are classified among membrane proteins, and as such are absent in the clarified lysate. Such distribution of isoelectric points among E. coli proteins opens the possibility to apply cation exchange in one-step purification of not only lysostaphin, but also other recombinant proteins characterized by high values of pI, using procedures described in this study.
This research was supported by funds Granted by the National Science Centre (NCN, Poland) based on the Decision No. DEC-2014/13/B/NZ1/00043 (to BW).
BW designed the study. AM and AJ performed the study. AM, AJ, GD and BW analyzed the results. AM, GD and BW wrote the manuscript. All authors revised the manuscript and agreed to be accountable for all aspects of the presented work.
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