Ján Krahulec

High level expression of human enteropeptidase light chain in Pichia pastoris.

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.

Increase in hyaluronic acid production by Streptococcus equi subsp. zooepidemicus strain deficient in β-glucuronidase in laboratory conditions

Streptococcus equi subsp. zooepidemicus is known to produce a hyaluronic acid capsule to resist the host immune defense. As the structure of the polysaccharide is identical to the one produced by humans, the bacteria S. equisubsp. zooepidemicusis used in biotechnological production of hyaluronic acid. In our laboratory we prepared mutated strains that are β-glucuronidase deficient. Comparing the wild-type strain, which is positive in β-glucuronidase activity, with the mutated strains named clone1 and clone2 in laboratory conditions, we observed that β-glucuronidase influences the production of hyaluronic acid considerably and the molecular weight of hyaluronan slightly. The production of hyaluronic acid by the mutated strains is higher by approximately 20% and the molecular weight is larger by about 2%. The significant increase in the production of hyaluronic acid and the slight increase in the molecular weight are probably caused by an absence of free β-glucuronic acid, due to its removal from the non-reducing termini of the polysaccharide by β-glucuronidase. The presence of free β-glucuronic acid would likely induce the expression of the β-glucuronic-acid-utilizing operon, which in turn would reflect into a misuse of energy in the glucose-rich media.

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

Influence of KfoG on capsular polysaccharide structure in Escherichia coli K4 strain

An enzyme KfoG with unknown function is coded by the gene kfoG. Gene kfoG belongs to genes from region 2, which are responsible for structure of capsular polysaccharide. Only two enzymes, KfoG and KfoC, coded by genes from region 2, have a glycosyltransferase motif. KfoC is the bifunctional enzyme, which is able to add both GalNAc and GlcUA on nascent polysaccharide, termed chondroitin polymerase. KfoG was predicted to be a fructosyltransferase. The gene that codes the KfoG enzyme was disrupted using homological recombination and absence of this gene was confirmed on both DNA and RNA levels. After disruption no structural changes have been observed, what indicates that fructose branching of the chondroitin backbone is not caused by enzymes, which are coded by genes from region 2 of the K4 capsular gene cluster.

Structure of the has operon promoter and the effect of mutations on the has promoter strength in Streptococcus equi subsp. zooepidemicus.

The purpose of this study is to determine the effect of the corresponding nucleotides from Streptococcus pyogenes on the has promoter strength in highly encapsulated strain S. equi subsp. zooepidemicus (SEZ) and detect an empowering mutations in SEZ. Eight different strains of SEZ carrying nucleotide mutations in the −73 to −38 region upstream of the has promoter were constructed. The significant activity decrease to 36–1% was observed after the introduction of mutations in the promoter region from −44 to −38 site. The exception was observed in mutation in −49 site when no significant decrease was observed. When nucleotides TTT were used in positions −73 the promoter became weaker, whereas no significant effect was observed after using nucleotides CCC (96%). Unfortunately, introduction of these mutations into chromosome SEZ has no empowering effect. Six strains, which carried nucleotide sequences of different lengths upstream from the transcription start of hasA promoter, were constructed to determine the minimum upstream region required for the maximum transcription efficiency of the has operon. No change of the activity of the has promoter constructs containing as few as 101 nucleotides upstream from the transcription start point was observed.

Native and denatured enzyme enterokinase determined by electrochemical methods

The aim of this work is to detect and distinguish native and denatured form of enzyme enteropeptidase. Cyclic voltammetry is usually the method of choice to be used in experiments with biomolecules or unknown chemical species. However, only hardly detectable differences between native and denatured form of enzyme enterokinase were observed using cyclic voltammetry. Therefore, chronopotentiometric stripping analysis was used for the characterization of protein conformation. Significant differences between the heights of peak H of native and denatured form of enterokinase were observed. Our experiments have proved that constant current chronopotentiometric peak H at mercury electrode is sensitive tool for the characterization of changes in protein conformation.Graphical abstract

Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains.

Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations.

Electrochemical determination of basic biochemical properties of enzyme enterokinase

Basic biochemical properties such as Michaelis constant (KM), turnover number (kcat), and the ratio between these two kinetic parameters of enzyme enteropeptidase were determined by electrochemical impedance spectroscopy. This method is developed for an easy, fast, and economic determination of mention enzyme characteristics and can be used for wide spectrum of biochemical systems. All values obtained by impedance measurements are compared and are in good agreement with those determined by traditional spectroscopic techniques and literature data. Therefore, one can suppose that electrochemical methods can be successful for such measurements also in the cases where usual ones (UV–Vis, fluorescence spectroscopy) are not able to determine these enzyme characteristics.Graphical abstract

Upstream regulatory regions controlling the expression of the Candida utilis maltase gene

Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. The current study presents preliminary characterization of the upstream regulatory regions controlling the carbon source-dependent expression of the C. utilis maltase gene. Cellobiose and soluble starch were recognised as inducers of maltase promoter, besides maltose. We successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in polyploid yeast C. utilis. Furthermore, the strength and minimal functional region of the promoter was evaluated by measuring β-galactosidase activity. Our results suggest, the qPCR was shown itself a very smart method for relatively easy characterization of the transformants and correlation of the expression levels with gene dosage.

Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli

The present study is focused on preparation of proper Escherichia coli expression system to ensure high yields of various modified precursors of human recombinant thrombin, a potential biopharmaceutical reagent. Two thrombin precursors, the smallest single-chain α-thrombin precursor prethrombin-2 and its shortened form prethrombin-2∆13, and their His-tagged forms were used. In order to determine the effect of the different lengths and amino acid compositions of affinity His-tag on the target protein expression level, a variety of the His-tag sequences were used. We found out that the protein expression efficiency was closely related to the codons used for encoding of amino acids of fusion histidine tag. Optimization of culture medium composition is another way to increase yield of the target protein. Suitable medium composition can ensure cell growth to high densities which is related to total yield of expressed protein. In this study, a new optimized complex medium for batch fermentation was developed. Addition of nutrients like a yeast extract and enzymatic casein hydrolysate to the defined medium components had a positive impact on protein expression, where relatively high expression level of the target protein from total amount of cellular proteins was achieved. Further, we have focused on trace element solution composition, and the optimized nickel and selenium concentrations were determined. Our results show that the composition of essential trace metal solution has a major impact not only on expression level, but it can also affect cell growth rate.