Recombinant production of lysyl endopeptidase (Lys-C) which is frequently used in proteomics is still challenging due to its complex structure. Herein, periplasmic expression and determining effective factors for recovery of the active enzyme were investigated. The codon-optimized Lys-C gene was cloned into pET26b (+) for periplasmic expression in E. coli Rosetta (DE3). The following parameters affecting expression level and activity of Lys-C were investigated including IPTG concentration (0.05-1 mM), cell density (OD: 0.45-0.8) at induction time, presence of reducing agents (glutathione or cysteine, 0-10 mM) in culture medium or periplasmic extraction buffers, and harvesting time (6 or 20 h). Lys-C was then purified by DEAE and Ni-NTA chromatography methods. The highest expression level was obtained at 0.05 mM IPTG (5.49 %), also 8 mM cysteine, induction at OD 0.45 and 6 h incubation increased enzyme activity to 23.5 %, 13.3 %, and 76.4 %, respectively. The enzyme activity of Lys-C in the presence of 4 mM glutathione and extraction buffers containing 2 mM 2-mercaptoethanol (2 ME) was 81.6 % higher than the condition without reducing agents. Also, 8 mM cysteine in the culture medium and 2 mM 2 ME in extraction increased the activity up to 29.7 %. Moreover, optimization of purification process enhanced the enzyme activity from 0.217 mU to 1.76 mU. Statistical analysis showed the examined parameters significantly affected enzyme activity (p < 0.05). The presence of the reducing agents in the culture medium and extraction buffers presumably improves the Lys-C folding and increases the enzyme activity.

Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0-10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba, Co, and Cu showed inhibitory effects on the activity of LysVPB, while Ca can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.

This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.

There has been a surge in the interest to utilize plants as hosts for producing vaccine antigens. In this study, we demonstrated the successful expression of the human parvovirus B19 (B19V) capsid protein (VP2) in Nicotiana benthamiana cells. The B19V VP1 and VP2 genes were cloned under the control of estrogen-inducible promoters and transiently expressed in N. benthamiana leaves using the agroinfiltration method. The addition of estrogen significantly boosted the expression of VP2. Furthermore, codon optimization of the VP2 sequence resulted in over a 30-fold increase in its expression compared with that of the wild-type. Analysis of negatively stained samples by sucrose density gradient ultracentrifugation and electron microscopy revealed that the expressed VP2 proteins formed spherical particles with diameters of approximately 20 nm. Immunostaining analysis of protoplasts derived from VP2-expressing N. benthamiana leaves indicated that VP2 signals were predominantly localized in the cytoplasm. These findings strongly suggested that B19V VP2 assembles and formed virus-like particles (VLPs) within the cytoplasm of N. benthamiana cells, presenting a promising method for producing B19V VLPs in plant systems.

Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen.

Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in k. These substrates with a short chain of 2-4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the -1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation.

Plant glutamate decarboxylase (GAD) is a Ca-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.

Na1.7 is a eukaryotic voltage-dependent Na channel (Na) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I-IV). It is involved in pain perception, and VSDs that differ significantly by Na subtype are targeted in the development of Na1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Na1.7 inhibitors, the chimera protein (NaAb/1.7VSD) of Na from prokaryotic Arcobacter butzleri (NaAb), in which extracellular loops of VSD were replaced with homologous loops from Na1.7, serves as an effective model. This is because NaAb harbors only one VSD and the biological activity of NaAb/1.7VSD was previously confirmed. To date, NaAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of NaAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Na1.7-specific peptide inhibitor. The results indicate that NaAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Na1.7 inhibitors using the PERISS screening method.

Survivin is an inhibitor of apoptosis, and expressed in a large number of cancers. As Survivin expression is very low in normal tissues, it assumes significance as a prominent target for tumor diagnosis, prognosis and developing anti-cancer therapies. We report development of a novel triple fusion protein for a prospective vaccine against Survivin in targeted cancer immunotherapy. A gene was synthesized by combining the nucleotides encoding human origin Survivin and heat-labile enterotoxin of Escherichia coli (LTB). Further, nucleotides corresponding to single chain variable fragment (scFv) of a monoclonal having affinity for DEC205 receptor present on dendritic cells, were also incorporated into the gene sequence. This complete gene was expressed to a triple fusion recombinant protein using a bacterial expression vector under the control of robust bacteriophage T7 promoter. The recombinant Survivin-LTB protein, with a size of approximately 60 kDa, was purified from the inclusion bodies using affinity based Ni-NTA columns. The purified protein was confirmed by the Western blot, and further characterized with circular dichroism, fluorescence spectroscopy and mass spectroscopy. This molecularly adjuvanted Survivin fusion protein designed to deliver to the dendritic cells for better antigen processing, elicited a stronger anti-Survivin immune response compared to Survivin protein alone. It can be an effective vaccine in active and passive immunotherapies for Survivin expressing cancer cells.

This study presents an exhaustive characterization of the enzymatic attributes and structural properties of trehalose-6-phosphate phosphatase (TPP) derived from Fusarium graminearum. Enzyme activity was evaluated through a meticulously designed enzymatic assay. The findings indicate that the molecular weight of the enzyme is approximately 99.8 kDa, with an optimal reaction temperature and pH of 40 °C and 6.5, respectively. Magnesium ions (Mg) markedly enhance the enzymatic activity, resulting in a specific activity of 1.795 U/μg. Kinetic analysis revealed a K value of 0.96 μmol/L and a V of 15.79 μmol/L/min. Subsequent computational analysis elucidated the three-dimensional architecture of the enzyme and identified the binding site for the substrate trehalose-6-phosphate (T6P). T6P was found to form hydrogen bonds with TPP at residues Lys754, Arg720, His665, Glu758, and Asn756. Additionally, hydrophobic interactions were observed between T6P and residues Phe802, Ile610, Asp801, Pro752, and Gly753. The binding energy calculated for the T6P-TPP complex stood at -5.7 kcal/mol.

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.

The recognition and characterization of gene-encoded nitric oxide synthase (NOS) from Exiguobacterium profundum are reported in this study. A new gene was sequenced and cloned from E. profundum and heterologously expressed in E. coli for functional identification, followed by protein purification using the His-tag. The stability and activity characteristics of the recombinant NOS were evaluated using different concentrations of IPTG at various time points. A band of approximately 42 kDa was observed by SDS-PAGE. The K value of NOS, calculated based on the Michaelis-Menten equation was 0.59 μmol/L. Additionally, homologous sequence alignment analysis indicated that the new NOS shared 80.48 % similarity with the same protein from Bacillus subtilis and Umezawaea. The construction of the NOS expression vector and the purification of the recombinant protein provide a foundation for further functional research and inhibitor development.

Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from Caldanaerobacter subterraneus subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3'-5' exonuclease, and 3'-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant TtAP in Escherichia coli with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in E. coli and constructed a fusion gene encoding TtAP with a 6His tag (TtAP-6His). TtAP-6His was put into vector pET-30a to form the expression vector pET-30a-TtAP-6His, and was then introduced into E. coli strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg).

Galactose oxidase, produced by fungi of the genus Fusarium, is an enzyme of great biotechnological importance. The gaoA gene has been recombinantly expressed in several hosts but has yet to be in Saccharomyces cerevisiae. This work aimed to express the Fusarium graminearum GaoA enzyme in S. cerevisiae. The full-length and truncated F. graminearum gaoA gene were subcloned into a yeast expression vector. The GaoA enzyme expression in S. cerevisiae was higher when the truncated gene, which codes for the mature form of the enzyme, was used. After purification of the expressed enzyme on a Sepharose 6B column, the obtained yield of the pure and active enzyme was 16.7 mg/L. The purified protein showed a K of 9.8 mM, lower than that of the wild-type enzyme, and a k/K of 2.9 × 10 Ms, higher than that of the wild-type enzyme. The expressed recombinant protein used several common substrates for galactose oxidase, such as galactose, raffinose, and 1,3-dihydroxyacetone dimer. In addition, it had increased activity on guar gum, lactose, and Arabic gum compared with the wild-type enzyme. The obtained enzyme´s characteristics are compatible with the galactose oxidase biotechnological applications.

Cryptomonad phycoerythrin 545 (PE545) is an important type of phycobiliprotein in basic research and technological innovations. Herein, we report a minimalistic hydrophobic chromatography method for its purification. High purity was achieved, with a purity ratio (A/A) of 13.66 and a recovery ratio of 78.63 %. Following SDS-PAGE, Coomassie Brilliant Blue staining revealed three bands at 9 kDa, 10 kDa, and 20 kDa, corresponding to α, α and β subunits. Multiple spectral characteristics were analyzed to ensure that optical activity was consistent with that of the natural protein. Absorption and fluorescence spectroscopies of purified PE545 displayed a strong absorption peak at 545 nm, a shoulder peak at 564 nm, and a fluorescence emission peak at 587 nm, which confirmed unchanged energy transfer properties. Furthermore, the structural and functional integrity, especially the existence of strongly coupled central chromophore pairs with excitation delocalization, was verified by circular dichroism and ultrafast absorption spectroscopy. From the studies of ultrafast absorption spectroscopy of excitation energy transfer (EET) of PE545, four decay components with lifetimes at 0.5 ps, 2.2 ps, 63 ps, and 3000 ps were obtained. In addition, the dynamics of these components confirmed the EET pathways from the central PEB chromophore pairs to the peripheral pigments and localized in the lowest state. Our work will be of considerable value for both fundamental research and applications of PE545.

Serum amyloid A1 (SAA1) is an apolipoprotein which is involved in amyloid A amyloidosis (AA) by forming fibrils. The process of fibrillation is still being explored and holds challenges in recombinant expression and purification of SAA1. This study deals with the preferable approach for the expression and purification of SAA1 which is normally toxic and unstable to express without using any fusion-tag. Complete soluble expression of SAA1 was obtained without the use of additional tag, in terrific broth, supplemented with 3 % ethanol at 30 °C. Soluble fraction of SAA1 was initially treated with salting-out using ammonium sulphate giving 1.5 M salt concentration to avoid SAA1 protein precipitation along with unwanted proteins. The soluble fraction of SAA1 after salting-out was purified by two individual chromatographic approaches: One anion exchange and second reverse phase chromatography. The yield of purified SAA1 was 3 times greater by anion exchange than reverse phase chromatography. MALDI-TOF analysis of purified SAA1 showed 11813 Da for intact protein and proteome analysis revealed greater than 90 % sequence coverage by MASCOT. The subunit interaction showed hexamer form at basic pH which was analyzed by size exclusion chromatography. The fibrillation activity of SAA1 was found to be 10-15 times higher in basic media at 43 °C than 37 °C. Our research demonstrates successful expression and purification of wild-type human recombinant SAA1. The cost-effective radical approach employed for purification of SAA1 is crucial for thorough protein characterization particularly, mechanisms of protein aggregation involved in amyloidosis.

Numerous proteins in nature strictly require oligomerization for their full activity. Moreover, the function of natural and artificial proteins can me adjusted by altering their oligomeric state, leading to development of biotechnologically-relevant biomacromolecules. Oligomerization scaffolds from natural sources and designed de novo enable shuffling the oligomeric state and valency of biomacromolecules. In this report we probed the scaffolding potential of the thermostable phenylacetic acid degradation protein acyl-CoA from Thermus thermophilus (TtPaaI). We designed and successfully produced the fusion protein between TtPaaI (scaffold) and galectin-7, a multifunctional lectin implicated in human diseases (ligand) and demonstrated that TtPaaI can serve as a framework for functional multivalent display of ligands.

Forkhead box protein J1 (FOXJ1) is the key transcriptional regulator during the conversion of mammalian primary cilium with a 9 + 0 architecture to the motile (9 + 2) one. The nucleotide sequences of the full-length and DNA-binding domain (DBD) of the open reading frame (ORF) were isolated and expressed into E. coli as 6xHis-tagged proteins. Upon induction, the DBD formed inclusion bodies that solubilized with 8 M urea. No induction of 6xHis-FOXJ1 protein was seen despite sub-cloning into several expression vectors and E. coli host strains. To improve induction and solubility, the 6xHis tag was substituted with Glutathione S-transferase (GST), and weak induction was seen in E. coli BL21(DE3). The GST-FOXJ1 showed anomalous migration on denaturing gel electrophoresis (AM-DRE), migrating at approximately 83 kDa instead of its calculated molecular weight (Mr) of 72.4 kDa. It was also unstable and led to degradation products. The 6xHis tag was substituted with Glutathione S-transferase (GST) to improve induction and solubility. Codon-optimization improved the induction, but the protein still showed AM-DRE and instability. It seemed that the recombinant protein was either toxic or posed a metabolic burden to the E. coli cells or, once produced was prone to degradation due mainly to the lack of post-translational modification (PTM). This process is required for some eukaryotic proteins after they are manufactured in the ribosomal factory. Both the purified recombinant proteins exhibited cysteine-induced oligomerization via the formation of disulphide bridges since this was reduced using dithiothreitol (DTT). Both were equally functional as these individually bound to an oligonucleotide, a consensus DNA-binding sequence for FOX proteins. Further, the recombinant polypeptides corresponding to the C-terminus and N-terminus show anomalies indicating that the highly acidic residues (known as polyacidic gel-shifting domains) in these polypeptides contribute to the AM-DRE. We demonstrate for the first time that the recombinant HsFOXJ1 and its DBD bind to DNA, its polyacidic gel-shifting domains are the reason for the AM-DRE, is unstable leading to degradation products, exhibits cysteine-induced oligomerization and harbours intrinsically disordered regions.

The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in Streptococcus pneumoniae, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed S. pneumoniae EgtU and purified it from E. coli BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization.

Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in Pichia pastoris by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (PpPDI) chaperon protein. Bos taurus chymosin was expressed under the control of a mutant ADH2 promoter. The clones containing 1-4 gene copy numbers of the chymosin were constructed using the in vitro cloning method, and the effect of chaperone protein on chymosin secretion was investigated. The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, PpPDI was over-expressed in the clones regulated with the ADH2 promoter. The over-expression of PDI gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL). The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and PpPDI genes were the optimum among the tested clones.

Acinetobacter baumannii (A. baumannii) is an opportunistic, Gram-negative human pathogen, which is predominantly found in hospital patients. Its antimicrobial resistance is escalating, leading to less efficient treatments, and an increasing interest in identifying new therapeutic drugs. Metals as antimicrobials are vital in healthcare and agriculture, and copper-containing surfaces are known to reduce microbial counts, also in clinical settings. Indeed, copper (Cu) is an essential element required for survival in all organisms from bacteria to humans, but nevertheless elevated levels are highly toxic for cells. Through different regulatory mechanisms, cells maintain Cu homeostasis, and ion channels and transporters are critical in this process. Precise understanding of such ion transport requires insight into the protein structures of the involved proteins, which will also provide information important for applied sciences. Considering the medical significance of A. baumannii and the possibility to exploit Cu to handle such infections, channels and transporters represent appealing targets. Here we approached the putative outer membrane CopB (Copper resistance protein B) from A. baumannii that is postulated to conduct Cu, with characterization of its structure and function as well as to enable rational drug-design. To this end, we demonstrate in this work procedures to produce purified sample and to recover diffracting protein crystals of CopB. The protein was overproduced in E. coli and membrane extracted in a range of detergents. The solubilized protein was subjected to crystallization, which yielded hits that scatter X-rays to low resolution. Our findings have the potential to pave the way for subsequent drug discovery.