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.

DNASynth: a computer program for assembly of artificial gene parts in decreasing temperature.

Artificial gene synthesis requires consideration of nucleotide sequence development as well as long DNA molecule assembly protocols. The nucleotide sequence of the molecule must meet many conditions including particular preferences of the host organism for certain codons, avoidance of specific regulatory subsequences, and a lack of secondary structures that inhibit expression. The chemical synthesis of DNA molecule has limitations in terms of strand length; thus, the creation of artificial genes requires the assembly of long DNA molecules from shorter fragments. In the approach presented, the algorithm and the computer program address both tasks: developing the optimal nucleotide sequence to encode a given peptide for a given host organism and determining the long DNA assembly protocol. These tasks are closely connected; a change in codon usage may lead to changes in the optimal assembly protocol, and the lack of a simple assembly protocol may be addressed by changing the nucleotide sequence. The computer program presented in this study was tested with real data from an experiment in a wet biological laboratory to synthesize a peptide. The benefit of the presented algorithm and its application is the shorter time, compared to polymerase cycling assembly, needed to produce a ready synthetic gene.

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.

New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system.

We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.