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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.

BackgroundThe most widespread, efficient prokaryotic protein-producing system is one where the T7 phage polymerase recognizes the T7 phage promoter (T7 p/p system). Unfortunately, in this system, target protein expression gradually declines and is often undetectable following 3 to 5 subcultures. Although a number of studies have attempted to stabilize the expression levels of the T7 p/p system, none has resolved the problem adequately and thus precludes the use of this system for the production of recombinant proteins on a large scale.ResultsWe created an expression cassette enabling stable, high-level expression in the T7p/p system. The cassette was tested with two different vector backbones and two target proteins. In all experiments, the expression system using the new cassette exhibited high and stable protein expression levels when compared to the traditional system.ConclusionsHerein, we describe a universal expression cassette that enables high-level, stable target protein expression in T7 RNA polymerase-based expression systems. We also present the successful use of this cassette as a novel expression platform and demonstrate its ability to overcome the main deficiency of the T7 p/p system. Thus, we provide a method for using the T7 p/p system on an industrial scale.

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.