Carratalá et al. Microbial Cell Factories (2023) 22:81 Microbial Cell Factories https://doi.org/10.1186/s12934-023-02081-7 RESEARCH Open Access Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N‑Lauroylsarcosine‑assisted affinity chromatography Jose Vicente Carratalá1,2,3†, Jan Atienza‑Garriga1,2,3†, Hèctor López‑Laguna1,2,3, Esther Vázquez1,2,3, Antonio Villaverde1,2,3*, Julieta M. Sánchez1,2,3,4* and Neus Ferrer‑Miralles1,2,3* Abstract 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 pro‑ teins 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 applica‑ tion 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-Lauroylsar‑ cosine. 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-Lauroylsarco‑ sine-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. Keywords Recombinant proteins, Difficult-to-purify proteins, E. coli, H6 tag, Detergent, Repurposing † 3 Jose Vicente Carratalá and Jan Atienza-Garriga contributed equally to this Departament de Genètica i de Microbiologia, Universitat Autònoma de work Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain 4 Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT) (CONICET- *Correspondence: Universidad Nacional de Córdoba), ICTA, FCEFyN, UNC., Av. Velez Sarsfield Antonio Villaverde 1611, X 5016GCA​ Córdoba, Argentina Antoni.villaverde@uab.cat Julieta M. Sánchez Julieta.sanchez@uab.cat Neus Ferrer‑Miralles Neus.Ferrer@uab.cat 1 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain 2 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER‑BBN), Instituto de Salud Carlos III, 08193 Cerdanyola del Vallès, Barcelona, Spain © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 2 of 14 Background solvent-exposed hydrophobic or cationic domains [21, The large-scale production of recombinant proteins has 22]. Although not deeply analyzed, failure of these pro- enabled their exploitation in a wide range of sectors such teins to be purified with sufficient efficiency (e.g., by as biomedicine and biotechnology, for diagnostics, ther- affinity chromatography) may be due to their tendency apy and vaccination [1–5], as well as molecular tools in to aggregate, even as soluble versions. Consequently, genetic engineering or catalysts in the biotech industry there is a steric sheltering of the purification tags or the [3, 6]. The production of recombinant proteins, espe- undesired acquisition of a sticky character that makes cially at large scale, suffers from important bottlenecks the polypeptide to interspecifically interact with other that minimize the yield of functional, usable products proteins. Despite the success in the application of N-L [7]. Among them, protein aggregation commonly occurs for IB protein recovery [20], this detergent had not been irrespective of the type of cell factory used for biofabrica- tested as a tool for improving the purification of soluble, tion [8, 9]. In bacteria, in which the recombinant proteins difficult-to-purify protein species. In this study and by are in general not secreted to the media, aggregation of using several model proteins, we have tested the capabil- the recombinant protein results in large cytoplasmic ity of N-L to favor the chromatographic purification of structures called inclusion bodies (IBs) [10]. Soluble complex recombinant proteins from Escherichia coli cell aggregates, probably intermediates in IB formation, also extracts, keeping their biological activity and avoiding abound [11–13], since aggregation of recombinant pro- traces of the detergent in the final product. According to teins is a complex event that involves a wide spectrum of the presented data, we propose the repurposing of N-L conformational conformers [14–17], ranging from prop- from an agent for the solubilization of protein aggregates erly folded versions to misfolded, amyloidal forms [16]. to an additive for the purification of soluble protein spe- Globally, the initial step in recombinant protein purifica- cies and the incorporation of this detergent as a highly tion is the separation, after cell lysis, of the soluble cell valuable tool for improved purification protocols to be fraction from the insoluble cell fraction. In this scenario, applied to the soluble cell fraction. the populations of recombinant protein that form IBs are retained in the insoluble cell fraction and therefore dis- Results carded from the protein purification process, that only GWH1-GFP-H6 is a modular protein (Fig. 1A, top) that involves soluble forms. For aggregation-prone proteins, contains GWH1, a cationic antimicrobial peptide (AMP) this fact represents an immediate loss of an important of clinical interest for the treatment of infectious diseases portion of the product that is straightforward excluded and in oncology [23]. The presence of the hexahistidine from the purification pipeline. (H6) at the carboxy terminus of the construct allows the However, proteins can also be recovered from IBs one-step purification of the protein by ­Ni2+-based affin- once these have been separated from the cell debris. In ity chromatography and, in addition, its self-assembling this regard, several procedures have been described for as regular homo-oligomers of around 10 nm. The oligo- the recovery of IB protein, based on either denaturing meric disposition of AMPs in multivalent nanoparticles or non-denaturing conditions. In the first, more conven- is highly desirable, as such format increases the local tional approach, the IB protein is completely denatured, concentration and effectiveness of the drug [24, 25]. The solubilized, and subjected to a refolding process aiming presence of GWH1 at the N-terminus of this construct at recovering the product in the native conformation and is a clear obstacle in the downstream of the protein, as with full functionality [18, 19]. This methodology shows the purity of the resulting product is much lower than variable and highly product-dependent success rates. For that of its counterpart GFP-H6 (Figure B, C). However, non-denaturing protein extraction, which is still emerg- when different detergents are added to the cell extracts, ing, standard protocols have been established that allow after mechanical cell lysis and before chromatography, the immediate release of IB proteins with full biological the purity of the products is enhanced from around 38% activity. This is achieved by using mild detergents like to more than 90% (Fig. 1C). While polysorbate 80 (P-80) N-Lauroylsarcosine (N-L, also known as sarkosyl) as had only a moderate effect (46% purity), sodium deoxy- solubilizing agents [20]. The success of this approach is cholate (S-D) and N-L were highly effective reagents. In based on the important amounts of recombinant proteins addition, P-80 also rendered a product that appeared to with native or native-like conformations contained in the be less proteolytically stable than alternative versions, IBs [17]. since a double protein band was observed in the gels Regardless of the generic optimization of protein (Fig. 1C). When checking the oligomeric architecture of recovery from large IB aggregates, many industrially or the proteins, S-D and N-L-managed products showed a clinically interesting proteins fall into the category of nanometric size compatible with previous observations, difficult-to-purify proteins, including those that have in a monodisperse peak (Fig. 1D). On the contrary, when Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 3 of 14 Fig. 1 Purification of GFP-H6 and GWH1-GFP-H6. A Modular architecture of GFP-H6 and GWH1-GFP-H6. Box sizes are only approximate. B Chromatographic profile for affinity chromatography of GFP-H6 and GWH1-GFP-H6 constructs present in the E. coli soluble cell fraction in the absence or presence of different detergents. Vertical dashed lines and numbers indicate the fraction selected for further analysis. C SDS-PAGE coupled to TGX stain-free gel Technology (Bio-Rad) of the protein fractions indicated through vertical lines in the panel B. D DLS size determination of GWH1-GFP-H6 purified in the presence of alternative detergents. Numbers indicate peak sizes. E Temperature-dependent DLS size data of GWH1-GFP-H6 purified in the presence of alternative detergents. F Circular Dichroism (CD) spectra of GWH1-GFP-H6 protein samples purified in the presence of alternative detergents. G GFP fluorescence spectrum of GWH1-GFP-H6 purified in the presence of alternative detergents P-80 was involved, a tendency to aggregate was suspected N-L as an additive in the cell extracts. These proteins due to the high polydispersion index showed by the pro- contained different AMPs at the N-terminus. In gen- tein material. Such tendency to aggregate was confirmed eral, AMPs show a cationic character expected to impair by increasing the temperature of the protein sample, fol- purification. Therefore, being these constructs models of lowed by its DLS analysis (Fig. 1E). Then, a temperature- difficult-to-purify proteins, they were well suited for the dependent increase in the size of the materials was only proposed analysis. As observed (Fig. 2B), the purity of observed when P-80 had been involved in their recovery. all these proteins dramatically improved when using the The higher stability of GWH1-GFP-H6 obtained with detergent-assisted chromatographic method for at least S-D and N-L was confirmed from the structural point of two-fold. Data are summarized in Fig. 2C. Again, to fur- view. The β structure of the protein revealed by circular ther discard specific links between the improved process dichroism was highly conserved in the three detergent and a particular protein domain, all these modular pro- strategies (Fig. 1F). However, the sample treated with teins were produced as new modular versions by substi- P-80 exhibited a lower intensity value in the whole GFP tuting the scaffold GFP by a central scaffold based on the emission fluorescence spectrum when compared to the murine interferon gamma (INFγ) (Fig. 3A). The purifica- samples treated with S-D and N-L (Fig. 1G). This result tion process of all these proteins was largely improved demonstrated a particular tertiary structure of GWH1- (Fig. 3B), and the final purity was dramatically enhanced, GFP-H6 obtained with P-80 that was in agreement with reaching almost 60-fold in the case of GWH1-INFγ-H6 the high size of protein oligomers and with the observed (Fig. 3C). instability (Fig. 1D, E). Of course, the presence of a detergent in the cell To test if the observed beneficial effect observed on extracts, even at low concentrations, might be a risk protein purification might be protein-specific, two addi- in a production process regarding the potential loss tional modular proteins based on GFP (Fig. 2A) were of conformation and functionalities of the target pro- produced in E. coli and purified as described above, using tein, which despite an improved yield could show a Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 4 of 14 Fig. 2 Comparative purification of cationic modular proteins based on GFP. A General architecture of the fusion proteins used here. Box sizes were only indicative. B Purity data summary. C and D Chromatographic profiles for affinity chromatography of PAD-GFP-H6 (C) and SPR2-GFP-H6 (D) from the E. coli soluble cell fraction in absence or presence of N-L in the cell extracts. Vertical dashed lines and numbers indicate the fraction selected for further analysis. The plots are sided by SDS-PAGE coupled to TGX stain free gel Technology (Bio-Rad) of protein fractions indicated in vertical lines Fig. 3 Comparative purification of cationic modular proteins based on INFγ. A General architecture of the fusion proteins used here. Box sizes were only indicative. B Purity data summary. C–E Chromatographic profiles for affinity chromatography of GWH1-INFγ-H6 (C), PAD-INFγ-H6 (D) and SPR2-INFγ-H6 from the E. coli soluble cell fraction in absence or presence of N-L in the cell extracts. Vertical dashed lines and numbers indicate the particular protein fraction selected for further analysis. The plots are sided by SDS-PAGE coupled to TGX stain free gel Technology (Bio-Rad) of protein fractions indicated by vertical lines Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 5 of 14 decreased biological activity. To check and to eventu- temperature of both protein samples determined from ally discard this possibility, a conventional, his-tagged E. the CMS vs temperature curve (Fig. 4D). Importantly, coli β-galactosidase was produced in recombinant form, and according to the absence of detectable conforma- and its enzymatic activity used as reporter to evalu- tional modifications, the specific activity of the enzyme ate a potential impairment of the conformational qual- remained unchanged when comparing both purification ity. Being a tetrameric enzyme with four active sites at protocols (Fig. 4G). the protein–protein interfaces, this enzyme is sensitive The improved purity of all the tested proteins (Figs. 1, to denaturing agents and any enzymatic loss should be 2, 3, 4) found upon detergent-assisted chromatographic indicative of a deleterious effect of the method. As in purification might be due, as presumed, to the hin- the case of previous tested proteins, N-L enhanced the drance of heterogeneous protein interactions but also to protein purity after affinity chromatography (Fig. 4A). a higher solvent-exposure of H6, expected to result in a When testing structural parameters we noted that both better binding of his-tagged polypeptides to N ­ i2+ in the protein samples, resulting from conventional purifica- columns. To test this hypothesis, the amount of recom- tion and from detergent-assisted purification, showed binant protein in the flow-through and wash eluates was indistinguishable properties. An overlap of the circu- measured for the six complex constructs produced in lar dichroism (Fig. 4B) and tryptophan fluorescence the study, using either the conventional purification pro- (Fig. 4C) spectra, and the DLS profiles (Fig. 4E) of both tocol or the detergent-assisted purification method. As detergent-treated and untreated β-galactosidase samples observed (Fig. 5A), all the tested proteins were retained confirmed that the secondary (Fig. 4B) and tertiary struc- by the columns with higher efficacies when the detergent tures (Fig. 4C and E) of the enzyme were preserved under was present in the extracts than when it was absent. Such the tested procedure. Moreover, the thermal stability of high affinity can account, by itself, the enhanced purity the protein until 50 °C was unaffected (Fig. 4D and F) and levels observed in the final product. Of course, since high there was not statistical difference between the unfolding retention in the affinity columns should not only result in Fig. 4 Purification of a recombinant E. coli β-galactosidase. A Comparative chromatographic profile of mAU signal at 280 nm for affinity chromatography of purification of β-gal-H6 with and without N-L. The corresponding SDS-PAGE coupled to TGX stain free gel Technology (Bio-Rad) of selected elution fractions is also shown. B Circular Dichroism (CD) spectra of β-gal-H6 protein samples purified with the N-L method C Tryptophan fluorescence spectrum of β-gal-H6 protein samples purified with N-L. D Center of Spectral Mass (CSM) of tryptophan fluorescence spectrum of β-gal-H6 samples purified with N-L, versus temperature. Inset: Tm values calculated from a sigmoidal model of CSM vs temperature. E DLS measurements of particle size distribution (by volume) of β-gal-H6 samples. F Thermal profile of β-gal-H6 protein size. G Comparative enzymatic activity of β-gal-H6 samples. Differences were not statistically different (p > 0.05) Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 6 of 14 Fig. 5 Binding of H6-tagged recombinant proteins to ­Ni2+ columns. A Western Blot (WB) of protein samples purified by the traditional protocol or assisted by N-L. For each protein, the binding efficiency of the proteins to the Ni-column is shown through the flow through (FT) and the wash (W) sample analyses. B General architecture of the fusion proteins used here to determine final yield. Box sizes were only indicative. C Summary of yield and purity data higher purity but in higher protein yields, this parameter of H6 in this situation than when the protein was stored was tested in the tagged proteins for which yield could be in absence of the surfactant, as modelled in Fig. 6B. precisely estimated when using the conventional purifica- Importantly, N-L was not detected in the final protein tion approach (that means, when the protocol rendered samples upon the dialysis steps (Appendix A), what was sufficient protein yields for their quantification) (Fig. 5B). of course relevant regarding the potential industry-ori- As expected, for the three tagged proteins yield, in addi- ented uses of the proposed protocol. tion to purity, was systematically improved, (Fig. 5C). The hypothesis that the presence of detergent leads Discussion to an increased solvent exposure of H6 in recombinant The final protein recovery yield in downstream recombi- proteins would be further supported if conformational nant protein production is a critical parameter for defin- changes could be detected in the protein upon incuba- ing the clinical or industrial applicability of promising tion with N-L at the working concentrations. This possi- proteins. Importantly, many of those valuable products bility was tested with the set of INFγ–based proteins and are discarded from industrial production and marketing the recombinant β–galactosidase. In all cases, the sets of because their low yield or unaffordable efforts required data demonstrated that in the presence of the detergent, for purification. Apart from the seminal protein fraction- a negligible or a slightly change in the CMS values cal- ing into soluble and insoluble versions, it is progressively culated from the Trp emission of the proteins occurred accepted from multiple independent observations that (Fig. 6A). As shown, the CMS values moved to different a recombinant protein produced in bacteria occurs in a or higher values in most of the samples (ΔCMS > 0) in continuum of conformations, and that soluble aggregates which N-L was present. This fact reflects moderate rear- are common in the cytoplasm or recombinant bacteria rangements of the protein structure mediated by N-L, [14–16, 26]. Taking this idea as a conceptual basis, the that might be compatible with a better solvent-exposure conformational spectrum of the soluble protein versions Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 7 of 14 Fig. 6 A Center of spectral mass (CMS) of the Trp emission (Appendix B) in the absence (control) or in the presence of N-L. These data were applied to calculate the ΔCMS = ­CMS+N-L – C ­ MScontrol and all the ΔCMS values were statistically different from zero (p < 0.05). B Speculative model of the mechanism of action of N-L, whose presence could place the his-tag in a more convenient way for its binding to the N ­ i2+ in the column. GWH1-GFP-H6 is shown at the top and GWH1-INFγ-H6 at the bottom. Colour codes are as it follows: yellow (antimicrobial peptide), red (his-tag) purple (scaffold protein) and green (N-L molecules) might have a differential impact on the purification effi- on the hypothesis that at least a fraction of difficult-to- cacies of such conformers, and at least partially, the purify protein populations might have conformational presence of these forms might also account for the occur- defects, the use of a mild detergent might improve their rence of difficult-to-purify proteins even if mostly pre- downstream. This would be especially relevant regarding sent in the soluble cell fraction. tag-based affinity chromatography, in which the affin- In this context, N-L is an anionic surfactant with mul- ity tag in the recombinant protein should be properly tiple applications in biotechnology and biomedicine, exposed to the solvent for a proper binding to the immo- including transdermal drug delivery [27–29], fabrica- bilized ligand [41–43]. We have proved here a dramatic tion of biomaterials and nanomaterials [30–32], struc- improvement in the column retention, final purity and tural protein analysis [33, 34], separation of co-purified recovery yield of several modular proteins (Figs. 1, 2, 3, 4, tag-less proteins from protein complexes [35]and mild 5), in which the cationic character of a fused N-terminus protein solubilization/extraction [36–38], among many domain clearly impairs its downstream (compare GFP- others. In fact, it was the first non-denaturing agent used H6 and GWH1-GFP-H6 in Fig. 1B and C). Although a in the solubilization of recombinant proteins from IBs compensation of charges between the cationic domain [39]. This early application in the handling of IBs fully and the anionic detergent and a consequent improve- supported the concept, developed much later [40], that ment of solubility cannot be discarded, the positive effect functional proteins could be recovered from insoluble that the surfactant shows on the purification of a conven- protein aggregates by mild detergents [20], in contrast to tional β-galactosidase (Fig. 4) indicates that such benefi- conventional and less technologically friendly denatur- cial impact has a transversal character irrespective of the ing/refolding-based protocols [18]. protein surface charge. In this line, N-L is among the most currently used Therefore, a model has been proposed to account for agents to favor non-denaturing protein extraction from the positive impact that N-L shows over H6-tagged pro- IBs, but its applicability as a generic assistant in the puri- tein absorption in ­Ni2+-based chromatography in which fication of soluble proteins had not been explored. Based the detergent slightly and reversibly unfolds the protein Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 8 of 14 (Fig. 6A), making H6 more solvent-exposed (Fig. 6B). This proteins with or without cationic domains and it does not is well supported by the moderate changes in the model alter their biological activities. Also, upon simple dialy- protein conformation reported here (Fig. 6A) as induced sis, the surfactant is removed in the final sample below by N-L. Interestingly, such modifications do not affect the detection limits of very fine analytical methods and the domain organization and probably involve local pro- far from any toxicologically relevant range. The structural tein zones. It must be noted that the enzymatic activity analyses of N-Lauroylsarcosine-exposed soluble protein of β-gal-H6, purified in absence or in presence of N-L, is indicate light conformational adjustments that might be indistinguishable (Fig. 4), and that the active sites of this compatible with a subtle conformational relaxation and a enzyme are located in the monomer–monomer inter- higher solvent-exposure of the purification tags. faces [44], making this enzyme sensitive to global confor- mational impacts. Also, in the line of potential obstacles for the use of N-L as a regular purification tool, the pres- Methods ence of detergent traces in the final product might pose Strains and genes severe health concerns. Indeed the presence of N-L and Three different Escherichia coli strains were employed related surfactants in intravenous, oral, transdermal and as hosts for the recombinant production of the different colorectal drug formulations as protein stabilizer inhib- proteins constructs, namely BL21 (DE3), BL21λ Codon its critical enzymes and raises several toxicological issues plus and Origami B (DE3). The modular proteins used [28, 45–47]. Regarding the handling of N-L-treated sam- here as models (Fig. 1A) followed the same architectonic ples, the working concentration used here (0.2%) is far principle. From N-terminus to C-terminus; an antimi- below the limit of 1–30%, which corresponds to Eye Irri- crobial peptide (AMP), a peptidic linker (GGSSRSS), a tation 2, H319, and below the limit of > 30%, which cor- scaffold protein (either GFP or INFγ) and a H6-Tag. In responds to Skin Irritation 2, H315. Furthermore, N-L GFP-H6, no AMP was present at the N-terminus. All is not classified as carcinogenic, mutagenic, or toxic for DNA sequences were synthetized by GeneArt (Waltham, reproduction, and is there are no evidence of chronic MA, USA) and codon-optimized for Escherichia coli. The toxicity, according to the 2nd ATP of Regulation (EC) No DNA segments encoding INFγ-H6 (mouse interferon 1272/2008 (CLP) and Directive 67/548/EEC (see https://​ gamma), GFP-H6, GWH1-GFP-H6, GWH1-INFγ-H6, echa.​europa.​eu/​web/​guest). Finally, N-L is not currently PaDBS1R1-GFP-H6 (abbrev. PAD-GFP-H6) [48], PAD- restricted by REACH. Regarding the final product, fine INFγ-H6, SRP2-GFP-H6 [49] and SRP2-INFγ-H6 were analytical methods have not detected detergent in the cloned into pET22b (­AmpR). On the other hand, a final samples obtained by the proposed procedure, with β-galactosidase-H6 (β-gal-H6) encoding gene was cloned an analytical detection limit of 1 ppm and a global detec- into pET26b ­(KanR). The different vectors containing the tion limit of 10 ppm for a ten-fold diluted original sample. synthetic genes were transformed by heat shock (42 °C This is achieved by conventional dialysis procedures that for 45 s) in chemically competent Escherichia coli BL21 might be further refined if convenient. Therefore, alto- (DE3) cells for the production INFγ-H6, GWH1-GFP- gether, the data presented here suggest the repurposing H6, GWH1- INFγ-H6, PAD-GFP-H6, PAD-INFγ-H6, of N-L from a solubilizator of IB proteins to its regular SRP2-GFP-H6 and SRP2-INFγ-H6, Escherichia coli use in the purification of difficult-to-purify soluble pro- BL21λ Codon plus for the production of β-gal-H6 or teins by affinity methods, as a valuable supporting agent Escherichia coli Origami B (DE3) for the production of able to improve the final yield and purity of the protein GFP-H6. Protein sequences are indicated in the Appen- up to more than 50-fold. dix C. Conclusions Gene expression and recombinant protein production N-Lauroylsarcosine is a detergent commonly used for the All E. coli cultures (1–2 L) were grown at 37 °C and mild solubilization of recombinant proteins from bacte- 250 rpm in LB broth with ampicillin at 100 μg/ml or kan- rial inclusion bodies. Here we have demonstrated that amycin at 34 μg/ml (only for β-gal-H6 production). E. coli the repurposing of this surfactant as a generic assistant Origami B (DE3) cultures were grown in LB with ampicil- in the affinity purification of His-tagged soluble proteins, lin at 100 μg/ml, kanamycin at 34 μg/ml and tetracycline upon its addition to the crude cell extracts, dramatically at 12.5 μg/ml. Once an optical density (­ OD600) of around improves the binding of H6-tagged proteins to ­ Ni2+. 0.5 was reached, isopropyl β-d-1-thiogalactopyranoside Consequently, both the yield and purity of the eluted (IPTG) was added at 0.1 mmol/L and the expression tem- material increases in more than one order of magnitude, perature set at 20 °C for and overnight culture. Cells were resulting in improved processes and in more industri- harvested by centrifugation (5000g for 15 min at 4 °C) ally appealing products. This approach is effective for and stored at − 80 °C. Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 9 of 14 Protein purification protocols Trp fluorescence For protein purification, the cell pellet was resuspended In proteins other than GFP we determined the trypto- in wash buffer (Buffer A = 40 mM Tris HCl (pH 8) / phan (Trp) fluorescence. The excitation and emission 500 mM NaCl) with the protease inhibitor complete slit were set at 5 nm. The excitation wavelength (λex) was EDTA-free (Roche). Bacterial cell lysis was performed 295 nm. Fluorescence emission spectra were acquired by high-pressure homogenization using the Avestin within a range from 300 to 500 nm. To evaluate the effect Emulsiflex C5 (ATA scientific). After the disruption, of N-L on the Trp emission, 0.2% of N-L was added to the cellular lysate was divided into aliquots of approxi- each protein sample. To analyse the thermal stability of mately 25 ml. To each one of these aliquots a specific proteins each spectrum was acquired in an increasing volume of a solution containing 40 mM Tris HCl (pH 8) temperature range (from 25 to 80ºC) and the tempera- and 500 mM NaCl, 2% N-L was added so that the final ture of unfolding (Tm) was calculated from the center of concentration of N-L was 0.2%. The sample contain- spectral mass (CSM) vs temperature curve as described ing the detergent was incubated at room temperature elsewhere [50]. For GFP versions, the excitation slit was with little agitation during 15–18 h. The next day, the set at 2.5 nm and the emission slit at 5 nm. The excita- soluble and insoluble fractions were separated by cen- tion wavelength (λex) was 488 nm. The fluorescence emis- trifugation (45 min, 15,000g at 4 °C) and the soluble sion spectra were acquired within a range from 500 to then through a 0.22 µm filter (Millex®-GP, Millipore fraction was filtrated firstly through a 0.45 µm filter and 600 nm. Express ® PES Membrane Filter Unit). Recombinant Circular dichroism proteins were purified in an Äkta Pure FPLC system Data were collected in a Jasco J-715 spectropolarimeter (GE Healthcare) by immobilized metal affinity chro- (JASCO, Oklahoma City, OK, USA) with a thermostatic matography (IMAC). After selective binding to a His- device by a Peltier system spectropolarimeter, using a Trap HP 5 ml column (GE Healthcare), proteins were 0.2 mm path length quartz cell. Each spectrum was an eluted by a linear gradient of elution buffer (40 mM average of seven scans. The protein concentration was Tris HCl (pH 8), 500 mM NaCl, 500 mM Imidazol and 0.15 mg/ml in each protein buffer. CD spectra were 0.2% N-L). The eluted fractions were analyzed through collected from 260–200 nm. Each final spectrum was Mini-PROTEAN TGX Stain-Free Gels and West- obtained from three replicas. Finally, we applied a nega- ern blot. The selected protein fractions were dialyzed tive exponential equation for the data smoothing. against sodium bicarbonate salt buffer (166 mmol/L ­NaCO3H and 333 mmol/L NaCl, pH 8.0) or bicarbonate Determination of β‑galactosidase enzymatic activity buffer (166 mmol/L ­NaCO3H, pH 8.0) and the final pro- β -Galactosidase activity was measured in PBS 1X by tein concentration was determined by NanoDrop One monitoring the colorimetric signal at 420 nm pro- Microvolume UV–Vis Spectrophotometer (Thermo duced by the degradation of an artificial substrate, Scientific). The regular purification protocol does not o-nitrophenyl-β-D-galactopyranoside (ONPG) [51]. include the presence of the detergent N-L and the over- night incubation at room temperature. Detection of N‑L in protein samples by mass spectrometry N-L in dialyzed protein samples was determined using mass spectrometry by the Chemical Analysis Service Dynamic light scattering (SAQ), a technological unit at the Universitat Autònoma The volume size distribution (expressed in nm) of all pro- de Barcelona (UAB). For that, an HPLC system was used tein candidates was determined by Dynamic Lights Scat- equipped with a DAD detector from Agilent Technologies tering (DLS) at 633 nm and both standard (25 °C) and and a micrOTOF-Q (time-of-flight) mass spectrometer increasing temperatures (from 4 to 50 °C) in a Zetasizer from Bruker Daltonics. Electrospray ionization in negative Advance Pro (Malvern Panalytical) using a ZEN2112 polarity was used to register the surfactant. 10 µL of sam- 3 mm quartz cuvette. Samples were measured at least ples were injected using water:acetonitrile (ACN) (1:1) at in triplicate, gaussian curves represented, and data 0.1 ml/min to the mass spectrometer. The parameters were expressed as mean ± standard error respectively. adjusted for sample injection from the liquid chromato- graph using Flow Injection Analysis (FIA) and detection in the mass spectrometer, without using any chromatographic Fluorescence measurements column, and taking a 20 ppm N-L standard to obtain maxi- Fluorescence spectra were obtained with a Cary Eclipse mum instrumental sensitivity. The mass spectrometer was spectrofluorometer (Agilent Technologies) with a quartz focused on low masses (m/z < 1000), given the molecular cell of 2 mm path length. Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 10 of 14 weight of the surfactant (m/z = 270.2), and the registrations using the default settings after introducing each primary were performed in negative polarity. To ensure that there FASTA sequence as query, respectively. Molecular graphics was no carryover between protein samples, the injection and analyses were performed with UCSF Chimera, devel- was changed to positive polarity, and the protein was then oped by the Resource for Biocomputing, Visualization, and injected. Data analysis indicated that there was no accumu- Informatics at the University of California, San Francisco, lation of protein in the instrument. Protein samples were with the support from NIH P41-GM103311 [54]. diluted 1:10 in water to reduce the carbonate concentra- tion and to make it compatible with the operational con- Statistical analysis ditions of the instrument. Control, N-L-enriched samples All statistical analyses were conducted in GraphPad Prism were prepared using the same procedure but adding N-L in version 8.0.0 for Windows (GraphPad Software, San Diego, the vial to 1 ppm. This method allows then the detection of CA, USA) with at least two independent replicates unless 1 ppm of surfactant per vial, that for ten-fold diluted sam- otherwise indicated. T tests to assess the differences in ples makes a detection limit of 10 ppm per protein sample. Tm values or in the specific enzymatic activity values were applied assuming unequal variances. All quantitative val- Protein design and Three‑Dimensional (3D) structure ues were expressed as mean ± standard error of the mean. prediction The significance of the statistical difference was included The 3D structures of the stable folded state of GWH1-GFP- in each experiment. A nonlinear regression analysis was H6 and GWH1-INFγ-H6 were predicted in silico using the developed to determine the melting temperature (Tm) of AlphaFold2 [52] algorithm integrated in ColabFold [53] and β-gal-H6 in both conditions from a sigmoidal model. Appendices Appendix A Analysis of N-L in dialyzed protein samples. Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 11 of 14 Superimposed Extracted Ion Chromatograms VNGHKFSVSGEGEGDATYGKLTLKFICTTG- (EICs) at m/z = 270.2 of a purified β-Gal-H6 sample KLPVPWPTLVTTLTYGVQCFSRYPDHMKRH- (grey) and an equivalent sample in presence of added DFFKSAMPEGYVQERTISFKDDGNYKTRAEVK- 1 ppm N-L (blue). F E G DT LV N R I E L KG I D F K E D G N I L G H K L E Y- NYNSHNVYITADKQKNGIKANFKIRHNIEDGS- VQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL- Appendix B SKDPNEKRDHMVLLEFVTAAGITHGMDELYHH- Tryptophan emission spectra of non GFP proteins HHHH (0.2 mg/ml) in the presence or absence of 0.2% N-L. Appendix C • GWH1-INFɣ-H6 MGYNYAKKLANLAKKFA- Amino acid sequences of the proteins used in the present NALWGGSSRSSHGTVIESLESLNNYFNSSGID- study. VEEKSLFLDIWRNWQKDGMKILQSQIISFYLR- LFEVLKDNQAISNNISVIESHLITTFFSNSKAK- • eGFP-H6 MSKGEELFTGVVPILVELDGDVNG- DAFMSIAKFEVNNPQVQRQAFNELIRVVQLL- HKFSVSGEGEGDATYGKLTLKFICTTGKLPVP- PESSLRKRKRSRCHHHHHH WPTLV TTLTYGVQ CFSRYPDHMKRHDFFK- • PaDBS1R1-GFP-H6 MPKILNKILGKILRLAAAFK- SAMPEGYVQERTISFKDDGNYKTRAEVKFEG- G GSSR SSSKGE E L F TG V V PILV E L D GDV N G- DTLVNRIELKGIDFKEDGNILGHKLEYNYNSH- HKFSVSGEGEGDATYGKLTLKFICTTGKLPVP- NVYITADKQKNGIKANFKIRHNIEDGSVQLAD- WPTLV TTLTYGVQ CFSRYPDHMKRHDFFK- HYQQNTPIGDGPVLLPDNHYLSTQSALSKDP- SAMPEGYVQERTISFKDDGNYKTRAEVKFEG- NEKRDHMVLLEFVTAAGITHGMDELYKHHH- DTLVNRIELKGIDFKEDGNILGHKLEYNYNSH- HHH NVYITADKQKNGIKANFKIRHNIEDGSVQLAD- • INFɣ-H6 MAQGQFFREIENLKEYFNASSPDVAK- HYQQNTPIGDGPVLLPDNHYLSTQSALSKDP- GGPLFSEILKNWKDESDKKIIQSQIVSFYFKLFEN- NEKRDHMVLLEF VTAAGITHGMDELYHHH- LKDNQVIQRSMDIIKQDMFQKFLNGSSEKLED- HHH FKKLIQIPVDDLQIQRKAINELIKVMNDLSPKSN- • PaDBS1R1-INFɣ-H6 MPKILNKILGKILRLAAAFK- LRKRKRSQNLFRGRRASTKHHHHHH GGSSRSSHGTVIESLESLNNYFNSSGIDVEEK- • GWH1-GFP-H6 MGYNYAKKLANLAKKFA- SLFLDIWRNWQKDGDMKILQSQIISFYLRLFE- NALWGGSSRSSSKGEELFTGVVPILVELDGD- VLKDNQAISNNISVIESHLITTFFSNSKAKDAFM- Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 12 of 14 SIAKFEVNNPQVQRQAFNELIRVVHQLLPESS- Acknowledgements Protein production was partially performed by the ICTS “NANBIOSIS”, more LRKRKRSRCHHHHHH specifically by the Protein Production Platform of CIBER in Bioengineering, • SRP2-GFP-H6 MGFALAGLARILCLWFREFSGF- Biomaterials & Nanomedicine (CIBER-BBN)/ IBB, at the UAB (http://​www.​nanbi​ FRRLNRRFAMRRRGGSSRSSSKGEELFTGVVPIL- osis.​es/​portf​olio/​u1-​prote​in-​produ​ction-​platf​orm-​ppp/). The authors also acknowledge the UAB scientific-technical services LLEB and SAQ (https://​ VELDGDVNGHKFSVSGEGEGDATYGKLTLK- www.​uab.​cat/​web/​resea​rch/​scien​tific-​techn​ical-​servi​ces/​scien​tific-​and-​techn​ FICTTGKLPVPWPTLVTTLTYGVQCFSRYPD- ical-​servi​ces--​13456​67278​676.​html) for the physicochemical characterization HMKRHDFFKSAMPEGYVQERTISFKDDGNYK- of the recombinant proteins. TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK- Author contributions L E YN YN SH N V YITADKQK N GI K ANFK I R H- JVC, JAG, HLL and JMS performed the experimental, analyzed the data and NIEDGSVQLADHYQQNTPIGDGPVLLPDNHYL- prepared the figures. EV, AV and NFM funded the study and designed and supervised the experimental. JVC, JAG and NFM designed the whole study STQSALSKDPNEKRDHMVLLEFVTAAGITHGM- and its concept. AV prepared the draft of the manuscript, that was revised and DELYHHHHHH edited by all the authors. All authors read and approved the final manuscript. • SRP2-INFɣ-H6 MGFALAGLARILCLWFREFSGF- Funding FRRLNRRFAMRRRGGSSRSSHGTVIESLESLNNY- The authors appreciate the financial support received for the develop‑ FNSSGIDVEEKSLFLDIWRNWQKDGDMKILQS- ment of multimeric recombinant drugs, from AEI (PID2020-116174RB-I00 QIISFYLRLFEVLKDNQAISNNISVIESHLITTFF- to A.V.; PID2019-105416RB-I00/AEI/10.13039/ 501100011033 and PDC2022- 133858-I00 to E.V.; PID2019-107298RB-C22 granted to N.F.-M) and AGAUR SNSKAKDAFMSIAKFEVNNPQVQRQAFNELIR- (SGR202100092 to A.V.). A.V. received an ICREA ACADEMIA award. JVCT is VVHQLLPESSLRKRKRSRCHHHHHH supported with a Margarita Salas grant for the training of young doctoral • β-gal-H6 GSHMLEDPVVLQRRDWENPGVTQLN- graduates (722713). J.A.-G. is supported by a predoctoral fellowship from the Ministerio de Universidades (FPU20/02260). HLL is supported by a predoc‑ RLAAHPPFASWRNSEEARTDRPSQQLRSLNGE- toral fellowship from AGAUR (2019 FI_B 00352). JMS is supported with a WRFAWFPAPEAVPESWLECDLPEADTVVVPSN- María Zambrano postdoctoral researcher contract (677904) from Ministerio WQMHGYDAPIYTNVTYPITVNPPFVPTENPT- de Universidades and European Union (“Financed by European Union-Next GenerationEU”). GC YSLTFNVDESWLQEGQTRIIFDGVNSAF- HLWCNGRWVGYG QDSRLPSEFDLSAFLR A- Availability of data and materials GENRLAVMVLRWSDGSYLEDQDMWRMSGI- The dataset supporting the conclusions of this article is available at https://​ doi.​org/​10.​34810/​data6​16 FRDVSLLHKPTTQISDFHVATRFNDDFSR AV- LEAEVQMCGELRDYLRVTVSLWQGETQVAS- GTAPFGGEIIDERGGYADRVTLRLNVENPKL- Declarations W S A E I P N LY R AV V E L H TA D G T L I E A E AC D - Ethics approval and consent to participate VGFREVRIENGLLLLNGKPLLIRGVNRHE- Not applicable. HHPLHGQVMDEQTMVQDILLMKQN- Consent for publication N F N AV R C S H Y P N H P LW Y T L C D R Y G LY V - Not applicable. V D E A N I E T H G M V P M N R LT D D P RW L PA M- Competing interests SERV TR MVQRDRNHPSVII WSLGNESGH- The authors declare no competing interests. G A N H D A LY R W I K S V D P S R P V Q Y E G G G A - DT TATDIIC PMYARVDED QPFPAVPKWSIK- Received: 2 February 2023 Accepted: 4 April 2023 KWLSLPGETRPLILCEYAHAAGNSLGGFAKY- WQAFRQYPRLQGGFVWDWVDQSLIKYDENG- NPWSAYGGDFGDTPNDRQFCMNGLVFADRT- PHPALTE AK HQ Q QFFQFRLSG QTIEV TSEY- References LFRHSDNELLHWMVALD GKPL ASGEVPLD- 1. Langer E, Rader R. Biopharmaceutical manufacturing: historical and VA P Q G KQ L I E L P E L P Q P E S AG Q LW LT V RV- future trends in titers, yields, and efficiency in commercial-scale bio‑ VQPNATAWSEAGHISAWQQWRLAENLSVTL- processing. BioProcessing J. 2015;13:47–54 2. Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, PAASHAIPHLTTSEMDFCIELGNKRWQFNRQS- Villaverde A. Recombinant pharmaceuticals from microbial cells: a 2015 GFLSQMWIGDKKQLLTPLRD QFTR APLDN- update. Microb Cell Fact. 2016;15:33. DIGVSEATRIDPNAWVERWKAAGHYQAEAAL- 3. Puetz J, Wurm FM. Recombinant proteins for industrial versus pharmaceutical purposes: a review of process and pricing. Processes. LQCTADTLADAVLITTAHAWQHQGKTLFISRK- 2019;7:476. TYRIDGSGQMAITVDVEVASDTPHPARIGLNC- 4. Pavlou AK, Reichert JM. Recombinant protein therapeutics—success QLAQVAERVNWLGLGPQENYPDRLTAACFDR- rates, market trends and values to 2010. Nat Biotechnol. 2004;22:1513–9. 5. de Pinho Favaro MT, Atienza-Garriga J, Martínez-Torró C, Parladé E, WDLPLSDMYTPYVFPSENGLRCGTRELNYG- Vázquez E, Corchero JL, et al. Recombinant vaccines in 2022: a perspec‑ PHQWRGDFQFNISRYSQQQLMETSHRHLL- tive from the cell factory. Microb Cell Fact. 2022;21:203. H AE EGT W L N I D GFHMGI G GDD S WSP S VS - 6. Deckers M, Deforce D, Fraiture MA, Roosens NHC. Genetically modified micro-organisms for industrial food enzyme production: an overview. AEFQLSAGRYHYQLVWCQKLGHHHHHH Foods. 2020;9:326. Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 13 of 14 7. Wingfield PT. Overview of the purification of recombinant proteins. Curr 30. Wang H, Wang JG, Zhou HJ, Liu YP, Sun PC, Chen TH. Facile fabrication Protoc Protein Sci. 2015. https://​doi.​org/​10.​1002/​04711​40864.​ps060​1s80. of noble metal nanoparticles encapsulated in hollow silica with radially 8. Corchero JL, Gasser B, Resina D, Smith W, Parrilli E, Vázquez F, et al. oriented mesopores: Multiple roles of the N-lauroylsarcosine sodium Unconventional microbial systems for the cost-efficient production of surfactant. Chem Commun. 2011;47:7680–2. high-quality protein therapeutics. Biotechnol Adv. 2013;31:140–53. 31. Titkov AI, Bulina NV, Ulihin AS, Shundrina IK, Karpova EV, Gerasimov EY, 9. Köszagová R, Nahálka J. Inclusion bodies in biotechnology. J Microbiol et al. N-Lauroylsarcosine capped silver nanoparticle based inks for flexible Biotechnol Food Sci. 2020;9:1191–6. electronics. J Mater Sci Mater Electron. 2017;28:2029. 10. Rinas U, Garcia-Fruitós E, Corchero JL, Vázquez E, Seras-Franzoso J, 32. Kurosaki T, Kishikawa R, Matsumoto M, Kodama Y, Hamamoto T, To H, et al. Villaverde A. Bacterial inclusion bodies: discovering their better half. Pulmonary gene delivery of hybrid vector, lipopolyplex containing N-lau‑ Trends Biochem Sci. 2017;42:726–37. roylsarcosine, via the systemic route. J Controlled Release. 2009;136:213. 11. Haacke A, Fendrich G, Ramage P, Geiser M. Chaperone over-expression 33. Thibeault J, Patrick J, Martin A, Ortiz-Perez B, Hill S, Zhang S, et al. Sarkosyl: in Escherichia coli: apparent increased yields of soluble recombinant a milder detergent than SDS for identifying proteins with moderately protein kinases are due mainly to soluble aggregates. Protein Expr high hyperstability using gel electrophoresis. Anal Biochem. 2019;571:21. Purif. 2009;64:185–93. 34. Mukherjee S, Dubois C, Perez K, Nisbet RM, Li QX, Varghese S, et al. 12. van der Henst C, Charlier C, Deghelt M, Wouters J, Matroule JY, Letes‑ Quantitative proteomics of detergent insoluble tangles derived from son JJ, et al. Overproduced Brucella abortus PdhS-mCherry forms Alzheimer’s disease brains. Alzheimers Dement. 2021;17:e055040. soluble aggregates in Escherichia coli, partially associating with mobile 35. Ukwaththage TO, Goodwin OY, Songok AC, Tafaro AM, Shen L, Mac‑ foci of IbpA-YFP. BMC Microbiol. 2010;10:248. naughtan MA. Purification of Tag-Free Chlamydia trachomatis Scc4 for 13. Fatima U, Singh B, Subramanian K, Guptasarma P. Insufficient (sub- structural studies using sarkosyl-assisted on-column complex dissocia‑ native) helix content in soluble/solid aggregates of recombinant tion. Biochemistry. 2019;58:4284–92. and engineered forms of IL-2 throws light on how aggregated IL-2 is 36. Balaban CL, Suárez CA, Boncompain CA, Peressutti-Bacci N, Ceccarelli EA, biologically active. Protein Journal. 2012;31. Morbidoni HR. Evaluation of factors influencing expression and extrac‑ 14. Martínez-Alonso M, González-Montalbán N, García-Fruitós E, Villaverde tion of recombinant bacteriophage endolysins in Escherichia coli. Microb A. The functional quality of soluble recombinant polypeptides pro‑ Cell Fact. 2022;21:40. duced in Escherichia coli is defined by a wide conformational spectrum. 37. Gifre-Renom L, Baltà-Foix R, Arís A, Garcia-Fruitós E. Nondenaturing solu‑ Appl Environ Microbiol. 2008;74:7431–3. bilization of inclusion bodies from lactic acid bacteria. Methods Mol Biol. 15. Vera A, González-Montalbán N, Arís A, Villaverde A. The conformational 2022;2406:389–400. quality of insoluble recombinant proteins is enhanced at low growth 38. Bader JA, Shoemaker CA, Klesius PH. Immune response induced by temperatures. Biotechnol Bioeng. 2007;96:1101. N-lauroylsarcosine extracted outer-membrane proteins of an isolate 16. Schrödel A, de Marco A. Characterization of the aggregates formed of Edwardsiella ictaluri in channel catfish. Fish Shellfish Immunol. during recombinant protein expression in bacteria. BMC Biochem. 2004;16:415–28. 2005;6:10. 39. Frankel S, Sohn R, Leinwand L. The use of sarkosyl in generating soluble 17. González-Montalbán N, García-Fruitós E, Villaverde A. Recombi‑ protein after bacterial expression. Proc Natl Acad Sci U S A. 1991;88:1192. nant protein solubility—does more mean better? Nat Biotechnol. 40. Peternel Š, Grdadolnik J, Gaberc-Porekar V, Komel R. Engineering inclusion 2007;25:718–20. bodies for non denaturing extraction of functional proteins. Microb Cell 18. Singh SM, Panda AK. Solubihzation and refolding of bacterial inclusion Fact. 2008;7:34. body proteins. J Biosci Bioeng. 2005;99:303. 41. Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purifi‑ 19. Vallejo LF, Rinas U. Strategies for the recovery of active proteins through cation. Curr Protoc Protein Sci. 2013. https://​doi.​org/​10.​1002/​04711​40864.​ refolding of bacterial inclusion body proteins. Microb Cell Fact. 2004;3:11. ps090​9s7. 20. Singhvi P, Saneja A, Srichandan S, Panda AK. Bacterial inclusion bodies: a 42. Mahmoodi S, Pourhassan-Moghaddam M, Wood DW, Majdi H, Zarghami treasure trove of bioactive proteins. Trends Biotechnol. 2020;38:474–86. N. Current affinity approaches for purification of recombinant proteins. 21. Zhang C-Y, Zhao S-Q, Zhang S-L, Luo L-H, Liu D-C, Ding W-H, et al. Data‑ Cogent Biol. 2019;5:1665406. base study on the expression and purification of membrane proteins. 43. Arnau J, Lauritzen C, Petersen GE, Pedersen J. Current strategies for the Protein Pept Lett. 2021;28:972–82. use of affinity tags and tag removal for the purification of recombinant 22. Antil M, Gouin SG, Gupta V. Truncation of C-terminal intrinsically disor‑ proteins. Protein Expr Purif. 2006;48:1–13. dered region of mycobacterial Rv1915 facilitates production of “difficult- 44. Matthews BW. The structure of E. coli β-galactosidase. C R Biol. to-purify” recombinant drug target. Front Bioeng Biotechnol. 2020;8:522. 2005;328:549–56. 23. Serna N, Céspedes MV, Sánchez-García L, Unzueta U, Sala R, Sánchez- 45. Bocci V, Naldini A, Corradeschi F, Lencioni E. Colorectal administration of Chardi A, et al. Peptide-based nanostructured materials with intrinsic human interferon-α. Int J Pharm. 1985;24:109–14. proapoptotic activities in ­CXCR4+solid tumors. Adv Funct Mater. 46. Whitehead K, Karr N, Mitragotri S. Discovery of synergistic permeation 2017;27:1700919. enhancers for oral drug delivery. J Controlled Release. 2008;128:128–33. 24. Carratalá JV, Serna N, Villaverde A, Vázquez E, Ferrer-Miralles N. Nanostruc‑ 47. Sattler J, Hesterberg R, Schmidt U, Crombach M, Lorenz W. Inhibition of tured antimicrobial peptides: the last push towards clinics. Biotechnol intestinal diamine oxidase by detergents: a problem for drug formula‑ Adv. 2020;44:107603. tions with water insoluble agents applied by the intravenous route? 25. Carratalá JV, Brouillette E, Serna N, Sánchez-Chardi A, Sánchez JM, Agents Actions. 1987;20:270–3. Villaverde A, et al. In vivo bactericidal efficacy of gwh1 antimicrobial 48. Irazazabal LN, Porto WF, Fensterseifer ICM, Alves ESF, Matos CO, Menezes peptide displayed on protein nanoparticles, a potential alternative to ACS, et al. Fast and potent bactericidal membrane lytic activity of antibiotics. Pharmaceutics. 2020;12:1217. PaDBS1R1, a novel cationic antimicrobial peptide. Biochim Biophys Acta 26. Gonzalez-Montalban N, Garcia-Fruitos E, Villaverde A. Recombinant Biomembr. 2019;1861:178–90. protein solubility—does more mean better? Nat Biotech. 2007;25:718–20. 49. Lützelberger M, Groß T, Käufer NF. Srp2, an SR protein family member 27. Kim YC, Park JH, Ludovice PJ, Prausnitz MR. Synergistic enhancement of fission yeast: In vivo characterization of its modular domains. Nucleic of skin permeability by N-lauroylsarcosine and ethanol. Int J Pharm. Acids Res. 1999;27:2618–26. 2008;352:129–38. 50. Carratalá JV, Cano-Garrido O, Sánchez J, Membrado C, Pérez E, Conchillo- 28. Lee H, Park J, Kim YC. Enhanced transdermal delivery with less irritation by Solé O, et al. Aggregation-prone peptides modulate activity of bovine magainin pore-forming peptide with a N-lauroylsarcosine and sorbitan interferon gamma released from naturally occurring protein nanoparti‑ monolaurate mixture. Drug Deliv Transl Res. 2018;8:54–63. cles. N Biotechnol. 2020;57:11–9. 29. Kováčik A, Kopečná M, Vávrová K. Permeation enhancers in transder‑ 51. Sánchez JM, López-Laguna H, Álamo P, Serna N, Sánchez-Chardi A, Nolan mal drug delivery: benefits and limitations. Expert Opin Drug Deliv. V, et al. Artificial Inclusion Bodies for Clinical Development. Advanced 2020;17:145–55. Science. 2020;7:1902420. Carratalá et al. Microbial Cell Factories (2023) 22:81 Page 14 of 14 52. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9. 53. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods. 2022;19:679–82. 54. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–12. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Ready to submit your research ? 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