A. Plucienniczak’s research while affiliated with Instytut Biotechnologii i Antybiotyków and other places

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.

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIGDM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIGDMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals.

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.

[This corrects the article DOI: 10.1371/journal.pone.0172008.].

Fig A in S1 Appendix SDS-PAGE showing (A) expression and (B) purification of rH5-E. coli. Fig B in S1 Appendix Antigenicity of rH5-E. coli. Table A in S1 Appendix Anti-H5 hemagglutinin antibodies used in this work. Table B in S1 Appendix Hemagglutination activity of rH5-E. coli. S1 Appendix Supplementary data on vaccine antigen. (DOC)

Background Cysteine proteinases of Fasciola hepatica are important candidates for vaccine antigens because of their role in fluke biology and host-parasite relationships. In our previous experiments, we found that a recombinant cysteine proteinase cloned from adult F. hepatica (CPFhW) can protect rats against liver fluke infections when it is administered intramuscularly or intranasally in the form of cDNA. We also observed considerable protection upon challenge following mucosal vaccination with inclusion bodies containing recombinant CPFhW produced in Escherichia coli. In this study, we explore oral vaccination, which may be the desired method of delivery and is potentially capable of preventing infections at the site of helminth entry. To provide antigen encapsulation and to protect the vaccine antigen from degradation in the intestinal tract, transgenic plant-based systems are used. Methodology In the present study, we aimed to evaluate the protective ability of mucosal vaccinations of 12-week-old rats with CPFhW produced in a transgenic-plant-based system. To avoid inducing tolerance and to maximise the immune response induced by oral immunisation, we used the hepatitis B virus (HBV) core protein (HBcAg) as a carrier. Animals were immunised with two doses of the antigen and challenged with 25 or 30 metacercariae of F. hepatica. Conclusions We obtained substantial protection after oral administration of the plant-produced hybrids of CPFhW and HBcAg. The highest level of protection (65.4%) was observed in animals immunised with transgenic plants expressing the mature CPFhW enzyme flanked by Gly-rich linkers and inserted into c/e1 epitope of truncated HBcAg. The immunised rats showed clear IgG1 and IgM responses to CPFhW for 4 consecutive weeks after the challenge.

Table A. Water intake changes in the subsequent days of tested insulins administration [multiple dose administration of insulin SR and insulin glargine (2 times a day; 2.5 and 5 U/kg b.w.)]. Table B. Food intake changes in the subsequent days of tested insulins administration [multiple dose administration of insulin SR and insulin glargine (2 times a day; 2.5 and 5 U/kg b.w.)]. Table C. Body weight changes in the subsequent days of tested insulins administration [multiple dose administration of insulin SR and insulin glargine (2 times a day; 2.5 and 5 U/kg b.w.)]. (DOCX)

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.

The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

Fig A in S1 Appendix SDS-PAGE showing (A) expression and (B) purification of rH5-E. coli. Fig B in S1 Appendix Antigenicity of rH5-E. coli. Table A in S1 Appendix Anti-H5 hemagglutinin antibodies used in this work. Table B in S1 Appendix Hemagglutination activity of rH5-E. coli. S1 Appendix Supplementary data on vaccine antigen. (DOC)