Recombinant human insulin: X. Simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing of Cys residues. The inclusion bodies were dissolved in 8 M urea with addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

OBJECTIVE--To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. DESIGN--Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. SETTING--Study in normal people at a diabetes research unit and a university department of medical physics. SUBJECTS--Seven healthy male volunteers aged 20-39 not receiving any other drugs. INTERVENTIONS--After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. END POINT--To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and more dramatic hypoglycaemic effect with the insulin analogue. CONCLUSIONS--The much faster absorption from subcutaneous tissue of the disubstituted monomeric insulin analogue compared with soluble insulin suggests that the analogue may be a potential candidate for rapid insulin delivery after subcutaneous bolus injection.     

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Recombinant human insulin IX. Investigation of factors,influencing the folding of fusion protein-S-sulfonates,biotechnological precursors of human insulin

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.     

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure.This structure causes highly efficient fusion protein folding.     

Recombinant human insulin V Optimization of the reversed-phase high-performance liquid chromatographic separation

The applicability of reversed-phase high-performance liquid chromatography (HPLC) to the analysis of the products of recombinant insulin was studied. The influence of several mobile phases in reversed-phase and ion-pair HPLC on selectivity, resolution and sensitivity was investigated. Optimum conditions for the separation of insulin-related proteins on commercial and laboratory-made supports were established by means of three-dimensional optimizations of selectivity and resolution as a function of pH and ionic strength (μ). A mechanism for the separation of proteins with a mobile phase containing a high salt concentration and a pH near the isoelectric point of proteins is proposed. The questions of scaling up are considered. The proposed techniques allow the analysis of the main impurities and ensures a high quality of active insulin production.     

Autoantibodies against human insulin.

Sera from 680 non-diabetic subjects with suspected autoimmune disease were screened for 13 different antibodies. Of the 582 sera found to contain these antibodies, nine bound insulin in an IgG specific enzyme linked immunosorbent assay (micro ELISA). Four of the sera bound human, porcine, and bovine insulins and five bound exclusively human insulin. "Cold" human, porcine, and bovine insulins each displaced, in a dose dependent manner, the four sera which bound all three insulins, but only human insulin displaced the remaining five, porcine and bovine insulins having little or no effect in concentrations up to 1000 U/1. These observations point to the existence of autoantibodies specifically against human insulin in some subjects with established autoimmunity.     

Recombinant human immunodeficiency viruses

Three types of recombinant human immunodeficiency virus have been found to circulate in Russia. The first type of the virus belongs to genotype gagAenvB, the second type belongs to genotype gagDenvG and the third type, to genotype gagAenvE. The virus of genotype gagAenvB circUlates in the population of drug addicts simultaneously with its "parent" viruses of genotypes gagAenvA and gagBenvB. The recombinant variant gagDenvG has African origin. The recombinant variant gagAenvE has probably been imported to Russia from South-East Asia.     

Recombinant human monoclonal antibodies

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed twoE. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked inE. coli:

1. Generation of a highly complex antibody repertoire;
2. Clonal selection procedures for library screening; and
3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.

     

Recombinant carbohydrate and selenomethionyl variants of human choriogonadotropin.

Recombinant human choriogonadotropin and selenomethionyl human choriogonadotropin (rhCG and SehCG) were expressed in baculovirus expression system by coinfection of SF9 insect cells by recombinant viruses, AcMNPV-hCG alpha and AcMNPV-hCG beta containing hCG alpha and hCG beta cDNAs. The expression efficiency of both rhCG and SehCG was quite high. The association of the alpha and beta subunits into a dimer was apparently complete since no detectable amount of rhCG beta was found in the rhCG eluate from the monoclonal hCG beta antibody immunoaffinity column. Both rhCG and SehCG preparations were homogeneous as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography. The apparent molecular mass of rhCG and SehCG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was about 38 kDa while under reducing conditions the heterodimer dissociated to yield beta and alpha subunits with molecular masses of 22.5 and 18 kDa, respectively. The carbohydrate analysis of rhCG showing the presence of 2.1, 3.3, 7.38, 4.2, and 27.8 residues of Fuc, GalNAC, GlcNAC, Gal, and Man, respectively, per mole of the hormone was consistent with the presence of 4 N-linked high mannose type carbohydrate hydrate and 4 O-linked simple carbohydrate chains, probably made up of Gal-GalNAC. Despite the altered glycosylation, rhCG demonstrated close similarity to the native urinary hCG in amino acid composition, receptor binding, and in its ability to stimulate cAMP and steroidogenesis. This indicates that there is no specificity of carbohydrate required for biological activity. Furthermore, it implies that the alteration from the complex to high mannose type carbohydrates in rhCG does not affect its proper folding. Finally, amino acid analysis of SehCG showed that 84% of methionine residues in rhCG were replaced by selenomethionine.     

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.     

The in vitro synthesized and processed human insulin receptor precursor binds insulin.

The cell-free examination of the human insulin receptor during biogenesis may provide a greater understanding of the elements that contribute to the acquisition of receptor function. The insulin receptor precursor components were produced in a cell-free system and the insulin binding ability of the [35S]methionine-labeled translation products was determined. The processed proreceptor represented by a 190 kDa band was retained on insulin-linked biotin-streptavidin agarose or an insulin column. The insulin binding 190 kDa band migrated slower than the non-binding 190 kDa band on SDS-PAGE which suggests that covalent modifications account for these differences. The trypsin-digested product of the 190 kDa proreceptor was also retained on insulin-linked biotin-streptavidin agarose, however the alpha-subunit precursor was retained on insulin agarose to a much lesser degree. We conclude that a significant fraction of the processed, in vitro translated insulin proreceptor acquires insulin binding ability.     

Recombinant live Salmonella spp. for human vaccination against heterologous pathogens

Live attenuated Salmonella spp. are promising candidates as oral vaccine delivery systems for heterologous antigens. Clinical trials have demonstrated that this approach is feasible for human vaccinations but further optimisation is necessary to obtain a better efficacy. Here, we discuss how existing clinical and pre-clinical data can be used to guide such optimisation efforts.