Elasticity Modification of Biomaterials Used in 3D Printing with an Elastin-Silk-like Recombinant Protein.

The recombinant structural protein described in this study was designed based on sequences derived from elastin and silk. Silk-elastin hybrid copolymers are characterized by high solubility while maintaining high product flexibility. The phase transition temperature from aqueous solution to hydrogel, as well as other physicochemical and mechanical properties of such particles, can differ significantly depending on the number of sequence repeats. We present a preliminary characterization of the EJ17zipR protein obtained in high yield in a prokaryotic expression system and efficiently purified via a multistep process. Its addition significantly improves biomaterial's rheological and mechanical properties, especially elasticity. As a result, EJ17zipR appears to be a promising component for bioinks designed to print spatially complex structures that positively influence both shape retention and the internal transport of body fluids. The results of biological studies indicate that the addition of the studied protein creates a favorable microenvironment for cell adhesion, growth, and migration.

Characterization of a Chimeric Resilin-Elastin Structural Protein Dedicated to 3D Bioprinting as a Bioink Component.

In this study we propose to use for bioprinting a bioink enriched with a recombinant RE15mR protein with a molecular weight of 26 kDa, containing functional sequences derived from resilin and elastin. The resulting protein also contains RGD sequences in its structure, as well as a metalloproteinase cleavage site, allowing positive interaction with the cells seeded on the construct and remodeling the structure of this protein in situ. The described protein is produced in a prokaryotic expression system using an E. coli bacterial strain and purified by a process using a unique combination of known methods not previously used for recombinant elastin-like proteins. The positive effect of RE15mR on the mechanical, physico-chemical, and biological properties of the print is shown in the attached results. The addition of RE15mR to the bioink resulted in improved mechanical and physicochemical properties and promoted the habitation of the prints by cells of the L-929 line.

Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment.

Single-chain variable fragment (scFv) antibodies are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. They possess unique and superior features compared to whole antibodies for immunotherapy of various carcinomas, including hematologic B-cell malignancies. In the presented study we obtained efficient production of the recombinant anti-CD22 scFv in Escherichia coli expression system. The active recombinant protein was successfully recovered from inclusion bodies. Assays were performed to assess the in vitro targeting properties and specificity of the obtained anti-CD22 scFv antibody in the CD22 positive and negative lymphoma cell lines.

Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions.

BACKGROUND: Gene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called "trivial overlapping", there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome's novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family. RESULTS: A single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR' protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR' have opposing functions - RepR is crucial for initiation of pIGRK replication, while RepR' is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression. CONCLUSIONS: Regulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Prime-Boost Vaccination With a Novel Hemagglutinin Protein Produced in Bacteria Induces Neutralizing Antibody Responses Against H5-Subtype Influenza Viruses in Commercial Chickens.

The highly pathogenic (HP) avian influenza virus (AIV), H5N1 and reassortant H5-subtype HPAIVs, H5N2, H5N6, and H5N8, cause high mortality in domestic birds, resulting in economic losses in the poultry industry. H5N1 and H5N6 also pose significant public health risks and H5N1 viruses are a permanent pandemic threat. To control HPAIVs, eukaryotic expression systems have traditionally been exploited to produce vaccines based on hemagglutinin (HA), a protective viral antigen. In contrast, we used a bacterial expression system to produce vaccine targeting the HA protein. A fragment of the HA ectodomain from H5N1, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. The resulting antigen, rH5-E. coli, was validated in terms of conformational integrity and oligomerization status. Previously, the protective efficacy of rH5-E. coli adjuvanted with aluminum hydroxide, has been positively verified by challenging the specific pathogen-free layer chickens with homologous and heterologous H5N1 HPAIVs. Protection was provided primarily by the H5 subtype-specific antibodies, as detected in the FluAC H5 test. The present studies were conducted to assess the performance of alum-adjuvanted rH5-E. coli in commercial birds. Broiler chickens were vaccinated twice with 25 µg of rH5-E. coli at 2- and 4-week intervals, while the layer chickens were vaccinated with 5- to 25-µg antigen doses at 4- and 6-week intervals. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition (HI) tests. Prime-boost immunizations with rH5-E. coli elicited H5 HA-specific antibodies in all the chickens tested. Two antigen doses administered at 4- or 6-week intervals were sufficient to induce neutralizing antibodies against H5-subtype HAs; however, they were ineffective when applied with a 2-week delay. In the layers, 80% to 100% of individuals developed antibodies that were active in the FluAC H5 and/or HI tests. A dose-sparing effect was seen when using the longer prime-boost interval. In the broiler chickens, 62.5% positivity was achieved in the FluAC H5 and/or HI tests. The trials confirmed the vaccine potential of rH5-E. coli and provided indications for anti-influenza vaccination with respect to the chicken type and immunization scheme.

Expression and purification of recombinant human insulin from E. coli 20 strain.

The number of people with diabetes is estimated to be over 370 million, in 2030 it will increase to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%). According to the estimates of the International Diabetes Federation, the percentage of patients in the adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of pharmacotherapy in cases of extremely high hyperglycemia. A new bacterial host strain (Escherichia coli 20) was obtained at the Institute of Biotechnology and Antibiotics and a new pIBAINS expression vector was constructed that provides greater efficiency in the production of recombinant human insulin. In the IBA Bioengineering Department, successful attempts were made to produce recombinant human insulin on a laboratory and quarter-technical scale, and several batches were performed on a semi-technical scale. The production process has been divided into several stages: 1. biosynthesis of insulin in the fermenter, 2. isolation, purification and dissolution of inclusion bodies, 3. protein renaturation, 4. enzymatic reaction with trypsin, 5. multi-stage purification of insulin using low-pressure and HPLC techniques. At each stage of insulin production, qualitative and quantitative analyses were performed to confirm identity and purity. In particular, the molecular weight of insulin, the amount of insulin and the content of protein impurities were studied. The results of these experiments are presented in this work.

Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters.

The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.

Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

pIGWZ12--A cryptic plasmid with a modular structure.

We studied the detailed structure of the cryptic plasmid pIGWZ12, which was isolated from an Escherichia coli strain. pIGWZ12 is composed of two structural modules of distinct evolutionary origin. The REP module, which contains all the features necessary for replication and stable maintenance in the bacterial cell, was assigned by genotyping to the IncF family. The MOB module, which is responsible for plasmid mobilization, shows significant homology to MOBQ modules from broad-host-range plasmids belonging to the RSF1010/R1162 family. We showed that iterons located in the origin of replication are the target for specific binding by the replication initiator protein RepApIGWZ12. Furthermore, we proved that the promoter for the repA gene overlaps with the iterons, and that the latter are the sole determinant of incompatibility. We performed a mutagenesis analysis of the MOBpIGWZ12 module and characterized the roles played by all identified genes (mobA and mobC), as well as the role played by oriT in mobilization. Finally, we showed that it was possible to remove the MOB module from pIGWZ12 without any loss in plasmid replication and stability. Furthermore, the MOBpIGWZ12 module was fully functional after subcloning into another plasmid. Therefore, pIGWZ12 is yet another example of modular structure in small cryptic plasmids.

Use of Ubp1 protease analog to produce recombinant human growth hormone in Escherichia coli.

BACKGROUND: Numerous bacterial human growth hormone (hGH) expression methods under conventional fermentation and induction conditions have been described. Despite significant progress made in this area over the past several years, production of recombinant hGH by using cellular expression systems still requires further optimization. Fusion of the ubiquitin (Ub) tag to the hGH protein allowed to increase of the overall efficiency of the biosynthesis and improve the protein stability. Ub is a protein composed of 76 amino acid residues with a molecular mass of 8.6 kDa, expressed in all eukaryotes. This protein is an element of the universal protein modification system, which does not occur in bacteria, and is a useful carrier for heterologous proteins obtained through expression in Escherichia coli. Purification of Ub-fusion proteins is easier than that of unconjugated recombinant proteins, and Ub can be removed by deubiquitinating proteases (DUBs or UBPs). RESULTS AND CONCLUSION: In the present study the UBPD2C protease, a stable UBP1 analog, was produced as a recombinant protein in E. coli and used for production of recombinant human growth hormone (rhGH). hGH was expressed as a fusion protein with Ub as a tag. Our findings show that the UBPD2C protease is very effective in removing the Ub moiety from recombinant Ub-fused hGH. The described approach enables obtaining a considerable yield of rhGH in a purity required for pharmaceutical products.

Expression of yeast deubiquitination enzyme UBP1 analogues in E. coli.

BACKGROUND: It has been shown that proteins fused to ubiquitin undergo greater expression in E. coli and are easier to purify and renaturate than nonhybrid foreign proteins. However, there is no commercial source of large quantities of specific deubiquitinating proteases. This is the reason why hybrid proteins containing ubiquitin at their N-end cannot be used in large scale biotechnological processes. RESULTS AND CONCLUSION: We have described the synthesis of the yeast deubiquination enzyme UBP1 muteins in E. coli. We have shown that an efficient overproduction of the enzyme in E. coli may be achieved after the introduction of several changes in the nucleotide sequence encoding UBP1. One of the conditions of an effective synthesis of the UBP1 muteins is the removal of the 5'-end sequence encoding the transmembrane region of the enzyme. The obtained variants of the enzyme may be successfully used for processing large amounts of hybrid proteins comprising ubiquitin or tagged ubiquitin at their N-ends.