Development of hybrid biomicroparticles: cellulose exposing functionalized fusion proteins.

BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected. CONCLUSIONS: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

An efficient method for the construction of artificial, concatemeric DNA, RNA and proteins with genetically programmed functions, using a novel, vector-enzymatic DNA fragment amplification-expression technology.

De novo designed bioactive molecules, such as DNA, RNA and peptides, are utilized in increasingly diverse scientific, industrial and biomedical applications. Concatemerization of designed DNA, RNA and peptides may improve their stability, bioactivity and allow for gradual release of the bioactive molecule at the intended destination. In this context, we developed a new method enabling the formation of DNA concatemers for the production of artificial, repetitive genes, encoding concatemeric RNAs and proteins of any nucleotide and amino-acid sequence. The technology recruits the Type IIS SapI restriction endonuclease (REase) for assembling DNA fragments in an ordered head-to-tail-orientation. Alternatively, other commercially available SapI isoschizomers can be used: LguI and thermostable BspQI. Four series of DNA vectors dedicated to the expression of newly formed, concatemeric open reading frames (ORFs), were designed and constructed to meet the technology needs. • Vector-enzymatic DNA fragment amplification technology. • Construction of DNA concatemers many times longer than those available with the use of current de novo gene synthesis methods. • Biosynthesis of protein tandem repeats with programmable function never seen in nature.

An alternative for proteinase K-heat-sensitive protease from fungus Onygena corvina for biotechnology: cloning, engineering, expression, characterization and special application for protein sequencing.

BACKGROUND: A neutral, heat-sensitive serine protease (NHSSP) originating from the feather-degrading fungus Onygena corvina (O. corvina) was described and defined as an alkaline serine protease of the subtilisin type S8 family, exhibiting an enzymatic activity at neutral pH. Generally, broad specificity proteases, such as proteinase K or trypsin, have found numerous applications in research and biotechnology. RESULTS: We report the cloning and expression in the yeast PichiaPink™ system, as well as purification, and characterization of the NHSSP. Recombinant, His6-tagged NHSSP was efficiently expressed from an optimized, synthetic gene and purified using a simple protocol based on ammonium sulfate fractionation and hydrophobic interaction chromatography. The enzyme shows atypical C-terminal processing, the coded preproprotein undergoes signal peptide removal and maturation through the clipping of a propeptide section and 10 amino acids (aa) from the C-terminus, including the His6-tag. The deletion variant has been constructed, devoid of the C-terminal ORF segment, thus eliminating the need for C-terminal processing. Both NHSSP variants exhibit very similar enzymatic characteristics. The purified enzymes were characterized to determine the optimal proteolytic conditions. We revealed that the mature NHSSP is reproducibly active over a wide pH range from neutral to mild acidic (pH of 5.0 to 8.5), with an optimum at pH 6.8, and at temperatures of 15 to 50 °C with an optimum at 38-42 °C. Interestingly, we demonstrated that the protease can be fully deactivated by a moderate increase in temperature of about 15 °C from the optimum to over 50 °C. The protease was partially sensitive to serine protease inhibitors, and not inhibited by chelating or reducing agents and detergents. SDS induced autolysis of NHSSP, which points to a high stimulation of its proteolytic activity. CONCLUSIONS: The NHSSP was produced as a recombinant protein with high efficiency. Compared to proteinase K, the most common serine protease used, NHSSP shows an approx. twofold higher specific activity. Protein sequencing can be a valuable technical application for the protease. The protein coverage is significantly higher in comparison to trypsin and reaches about 84-100% for ß-lactoglobulin (BLG), antibody (mAb) light and heavy chains. Furthermore, the option to perform digestions at neutral to slightly acidic pH-values down to pH 5.0 avoids modification of peptides, e.g. due to deamidation.

Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method.

Applications of bioactive peptides and polypeptides are emerging in areas such as drug development and drug delivery systems. These compounds are bioactive, biocompatible and represent a wide range of chemical properties, enabling further adjustments of obtained biomaterials. However, delivering large quantities of peptide derivatives is still challenging. Several methods have been developed for the production of concatemers - multiple copies of the desired protein segments. We have presented an efficient method for the production of peptides of desired length, expressed from concatemeric Open Reading Frame. The method employs specific amplification-expression DNA vectors. The main methodological approaches are described by Skowron et al., 2020 [1]. As an illustration of the demonstrated method's utility, an epitope from the S protein of Hepatitis B virus (HBV) was amplified. Additionally, peptides, showing potentially pro-regenerative properties, derived from the angiopoietin-related growth factor (AGF) were designed and amplified. Here we present a dataset including: (i) detailed protocols for the purification of HBV and AGF - derived polyepitopic protein concatemers, (ii) sequences of the designed primers, vectors and recombinant constructs, (iii) data on cytotoxicity, immunogenicity and stability of AGF-derived polypeptides.

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications.

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.

Randomized DNA libraries construction tool: a new 3-bp 'frequent cutter' TthHB27I/sinefungin endonuclease with chemically-induced specificity.

BACKGROUND: Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. RESULTS: In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5'-CAARCA-3' is converted into a combined 3.2-3.0-bp 'site' or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. CONCLUSIONS: In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA.

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.

The third restriction-modification system from Thermus aquaticus YT-1: solving the riddle of two TaqII specificities.

Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5'-GACCGA-3' and 5'-CACCCA-3'. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5'-GACCGA-3' sequence only. Here, we report the discovery, isolation, and characterization of TaqIII, the third R-M system from T. aquaticus YT-1. TaqIII is a 1101-aa Type IIC/IIL enzyme and recognizes the 5'-CACCCA-3' sequence previously attributed to TaqII. The cleavage site is 11/9 nucleotides downstream of the A residue. The enzyme exhibits striking biochemical similarity to TaqII. The 93% identity between their aa sequences suggests that they have a common evolutionary origin. The genes are located on two separate plasmids, and are probably paralogs or pseudoparalogs. Putative positions and aa that specify DNA recognition were identified and recognition motifs for 6 uncharacterized Thermus-family enzymes were predicted.

Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I.

The Thermus sp. family of bifunctional type IIS/IIG/IIC restriction endonucleases (REase)-methyltransferases (MTase) comprises thermo-stable TaqII, TspGWI, TspDTI, TsoI, Tth111II/TthHB27I enzymes as well as a number of putative enzymes/open reading frames (ORFs). All of the family members share properties including a large protein size (ca. 120kDa), amino acid (aa) sequence homologies, enzymatic activity modulation by S-adenosylmethionine (SAM), recognition of similar asymmetric cognate DNA sites and cleavage at a distance of 11/9 nt. Analysis of the enzyme aa sequences and domain/motif organisation led to further Thermus sp. family division into the TspDTI and TspGWI subfamilies. The latter exhibits an unprecedented phenomenon of DNA recognition change upon substitution of SAM by its analogue, sinefungin (SIN), towards a very frequent DNA cleavage. We report cloning in Escherichia coli (E. coli), using a two-stage procedure and a putative tthHB27IRM gene, detected by bioinformatics analysis of the Thermus thermophilus HB27 (T. thermophilus) genome. The functionality of a 3366 base pair (bp)-/1121 aa-long, high GC content ORF was validated experimentally through the expression in E. coli. Protein features corroborated with the reclassification of TthHB27I into the TspDTI subfamily, which manifested in terms of aa-sequence/motif homologies and insensitivity to SIN-induced specificity shift. However, both SAM and SIN stimulated the REase DNA cleavage activity by at least 16-32 times; the highest was observed for the Thermus sp. family. The availability of TthHB27I and the need to include SAM or SIN in the reaction in order to convert the enzyme from "hibernation" status to efficient DNA cleavage is of practical significance in molecular biotechnology, extending the palette of available REase specificities.

A new prototype IIS/IIC/IIG endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus, recognising 5'-TARCCA(N11/9)-3' sequences.

The Thermus sp. family of IIS/IIG/IIC enzymes includes the thermostable, bifunctional, fused restriction endonuclease (REase)-methyltransferases (MTase): TaqII, Tth111II/TthHB27I, TspGWI, TspDTI and TsoI. The enzymes are large proteins (approximately 120kDa), their enzymatic activities are affected by S-adenosylmethionine (SAM), they recognise similar asymmetric cognate sites and cleave at a distance of 11/9 nucleotides (nt). The enzymes exhibit similarities of their amino acid (aa) sequences and DNA catalytic motifs. Thermus sp. enzymes are an example of functional aa sequence homologies among REases recognising different, yet related DNA sequences. The family consists of TspGWI- and TspDTI-subfamilies. TsoI appears to be a non-identical 'triplet', related to TspDTI and Tth111II/TthHB27I. The discovery of TsoI, purified from Thermus scotoductus, is described. This prototype, displaying a novel specificity, which was determined by: (i) cleavage of a reference plasmid and bacteriophage DNA, (ii) cleavage of custom PCR DNA substrates, (iii) run-off sequencing of cleavage products and (iv) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with TsoI. The enzyme recognises a degenerated 5'-TARCCA-3' sequence, whereas DNA strands are cut 11/9 nt downstream. The discovery of the TsoI prototype is of practical importance in biotechnology, as it extends the palette of cleavage specificities for gene cloning.