A new pH-responsive peptide tag for protein purification.

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH.

Insulin chains as efficient fusion tags for prokaryotic expression of short peptides.

Insulin chains are usually expressed in Escherichia coli as fusion proteins with different tags, including various low molecular weight peptide tags. The objective of this study was to determine if insulin chains could facilitate the recombinant expression of other target proteins, with an emphasis on low molecular weight peptides. A series of short peptides were fused to mini-proinsulin, chain B or chain A, and induced for expression in Escherichia coli. All the tested peptides including glucagon-like peptide 1 (GLP-1), a C-terminal extended GLP-1, oxyntomodulin, enfuvirtide, linaclotide, and an unstructured artificial peptide were expressed with reasonable yields, identified by Tricine-SDS-PAGE and immunoblotting. All recombinant products were expressed in inclusion bodies. The effective accumulation of products was largely attributed to the insoluble expression induced by fusion with insulin chains, and was confirmed by the fusion expression of transthyretin. Insulin chains thus show promise as efficient fusion tags for mass production of heterologous peptides in prokaryotes.

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry.

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

Association of newly synthesized islet prohormones with intracellular membranes.

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.

[Optimization of the industrial production of the recombinant precursor of human insulin].

Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.

Equilibrium folding of porcine insulin precursor in the presence of redox buffer: implications for the common intermediates shared by its unfolding/ refolding processes.

We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.

Contribution of the conserved A16Leu to insulin foldability.

Contributions of the evolutionarily conserved A16Leu and B17Leu to insulin foldability were characterized by evaluating folding properties of single-chain insulin analogs. The results showed A16Leu had much more significant effects on the foldability of insulin than B17Leu.

Familial hyperproinsulinemia. Two cohorts secreting indistinguishable type II intermediates of proinsulin conversion.

Familial hyperproinsulinemia, a hereditary syndrome in which individuals secrete high amounts of 9,000-mol wt proinsulin-like material, has been identified in two unrelated cohorts. Separate analysis of the material from each of the two cohorts had suggested that the proinsulin-like peptide was a conversion intermediate in which the C-peptide remained attached to the insulin B-chain in one case, whereas it was a conversion intermediate in which the C-peptide remained attached to the insulin A-chain in the other. To reinvestigate this apparent discrepancy, we have now used chemical, biochemical, immunochemical, and physical techniques to compare in parallel the structures of the immunoaffinity chromatography-purified, proinsulin-like peptides isolated from the serum of members of both families. Our results show that affected individuals in both cohorts secrete two-chained intermediates of proinsulin conversion in which the COOH-terminus of the C-peptide is extended by the insulin A-chain and from which the insulin B-chain is released by oxidative sulfitolysis. Analysis of the conversion intermediates by reverse-phase high-performance liquid chromatography using two different buffer systems showed that the proinsulin-related peptides from both families elute at a single position very near that of the normal intermediate des-Arg31, Arg32-proinsulin. Further, treatment of these peptides with acetic anhydride prevented trypsin-catalyzed cleavage of the C-peptide from the insulin A-chain, a result demonstrating the presence of Lys64 and the absence of Arg65 in both abnormal forms. We conclude that individuals from both cohorts with familial hyperproinsulinemia secret very similar or identical intermediates of proinsulin conversion in which the C-peptide remains attached to the insulin A chain and in which Arg65 has been replaced by another amino acid residue.

The interactions of proinsulin with insulin receptors on the plasma membrane of the liver.

The interactions of proinsulin with the insulin-specific receptors were investigated in purified rat liver plasma membranes. These studies were designed to characterize the binding of proinsulin to the insulin receptors, to search for proinsulin-specific receptor sites, and to examine the possibility of proinsulin conversion at the insulin receptor site. Proinsulin was only 3-5% as potent as insulin in binding to insulin receptors. Proinsulin reacted with all of the insulin-specific receptors, and direct binding studies of [(125)I]porcine proinsulin and [(125)I]rat proinsulin did not reveal proinsulin-specific receptor sites other than the insulin receptors in rat liver membranes. Quantitative data derived from steady-state and transient-state comparative binding studies of both [(125)I]proinsulin and [(125)I]insulin indicated that a 20-fold lower association rate constant essentially accounts for the reduced affinity of proinsulin for the insulin receptors. The possibility of proinsulin conversion at the insulin receptor sites was investigated. Material recovered from the membranes upon dissociation of the proinsulin-receptor complex was intact proinsulin and did not exhibit any conversion by a variety of analytical methods. These results indicate that the lower affinity of proinsulin for the insulin receptor in the liver is an intrinsic property of the proinsulin molecule. The lower uptake of proinsulin by the insulin receptor represents, in addition to a slower degradation of the prohormone, a further mechanism by which proinsulin exerts prolonged, albeit reduced, action in vivo.

Affinity chromatography matures as bioinformatic and combinatorial tools develop.

Affinity chromatography has the reputation of a more expensive and less robust than other types of liquid chromatography. Furthermore, the technique is considered to stand a modest chance of large-scale purification of proteinaceous pharmaceuticals. This perception is changing because of the pressure for quality protein therapeutics, and the realization that higher returns can be expected when ensuring fewer purification steps and increased product recovery. These developments necessitated a rethinking of the protein purification processes and restored the interest for affinity chromatography. This liquid chromatography technique is designed to offer high specificity, being able to safely guide protein manufactures to successfully cope with the aforementioned challenges. Affinity ligands are distinguished into synthetic and biological. These can be generated by rational design or selected from ligand libraries. Synthetic ligands are generated by three methods. The rational method features the functional approach and the structural template approach. The combinatorial method relies on the selection of ligands from a library of synthetic ligands synthesized randomly. The combined method employs both methods, that is, the ligand is selected from an intentionally biased library based on a rationally designed ligand. Biological ligands are selected by employing high-throughput biological techniques, e.g. phage- and ribosome-display for peptide and microprotein ligands, in addition to SELEX for oligonucleotide ligands. Synthetic mimodyes and chimaeric dye-ligands are usually designed by rational approaches and comprise a chloro-triazinlyl scaffold. The latter substituted with various amino acids, carbocyclic, and heterocyclic groups, generates libraries from which synthetic ligands can be selected. A 'lead' compound may help to generating a 'focused' or 'biased' library. This can be designed by various approaches, e.g.: (i) using a natural ligand-protein complex as a template; (ii) applying the principle of complementarity to exposed residues of the protein structure; and (iii) mimicking directly a natural biological recognition interaction. Affinity ligands, based on the peptide structure, can be peptides, peptide-mimetic derivatives (<30 monomers) and microproteins (e.g. 25-200 monomers). Microprotein ligands are selected from biological libraries constructed of variegated protein domains, e.g. minibody, Kunitz, tendamist, cellulose-binding domain, scFv, Cytb562, zinc-finger, SpA-analogue (Z-domain).

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

On the preparation and characterization of standard materials for natural human proinsulin and C-peptide.

The preparation and characterization of intact native human proinsulin and C-peptide to serve as reference standards in immunologic and biologic assays is described. The major difficulty encountered in the preparation of human proinsulin from crystalline human insulin is the rather low yield (0.08 per cent), which may be due to postmortem autolysis in the pancreatic specimens. Under such circumstances, minor degrees of contamination of batches by fresh pancreases from other species, such as cows or pigs, may be magnified considerably. Such contamination can be readily detected with various currently available proinsulin- or C-peptide-specific antisera. Autolysis before or during preparation may also affect the yields and quality of human C-peptide preparations. Some of these C-peptide by-products are described, along with methods for their purification and characterization.

Metabolism of des(64,65)-human proinsulin in the rat. Evidence for the proteolytic processing to insulin.

The metabolism of des(64,65)-human proinsulin was examined in rats after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.

Relative hypersecretion of proinsulin in rat model of NIDDM.

The plasma ratio of proinsulin to insulin is raised in individuals with non-insulin-dependent diabetes mellitus (NIDDM). Increased secretion of proinsulin relative to insulin is thought to be the cause, although differential changes in clearance have not been ruled out. This study was conducted in a rat model of NIDDM, 90% pancreatectomized (Px) rats, to investigate the pathophysiology of this observation. Proinsulin storage and secretion were assessed with high-performance liquid chromatography separation of the insulins and the proinsulins, followed by quantification of the peaks by insulin radioimmunoassay. In Px rats, the relative proportion of proinsulin in pancreas extracts was twice that of control (sham-operated) rats (15.6 +/- 1.4 vs. 8.3 +/- 1.4%, P less than 0.01). Samples obtained from the portal vein during in vitro pancreas perfusion also had an elevated proinsulin fraction (Px, 10.3 +/- 3.0; sham, 3.0 +/- 0.6%; P less than 0.006). In summary, 90% Px rats share many pathophysiological features with NIDDM, including loss of normal proinsulin homeostasis. Our results suggest that chronic hyperglycemia causes an intrinsic change in beta-cells that is characterized by the increased storage and secretion of proinsulin.

Identification of insulin variants in patients with hyperinsulinemia by reversed-phase, high-performance liquid chromatography.

We have characterized the molecular forms of circulating insulins in patients with hyperinsulinemia of diverse etiology. We have also compared the efficacy of various chromatographic conditions using reversed-phase (RP) HPLC. Using 0.2% trifluoroacetic acid (TFA) and triethylamine (TEA) with acetonitrile as the organic modifier, at an elution rate of 0.17%/min, porcine, bovine, and human insulins could be easily separated as well as abnormal insulins in the plasma of a patient (J.R.) with hyperinsulinemia of unknown etiology. When the reversed-phase C18 column was changed and a gradient of 0.33%/min was used, the abnormal insulin in patient J.R. could not be separated. By changing the solvent system to acetonitrile and isopropanol (vol:vol, 3:1) containing 0.1% TFA, omitting the TEA, and using a gentle gradient of 0.1%/min, various semisynthetic analogues of human insulin could be easily separated and the abnormal insulin could be identified in the plasma of the patient J.R. Abnormal insulin was also found in a patient with MEN-I, but in contrast, the insulins in eight patients with benign sporadic insulinomas appeared to be normal. These results suggest that certain hyperinsulinemic states may be associated with an abnormal insulin and that RP-HPLC is useful for identification of insulin variants in the circulation. However, the conditions of RP-HPLC may be critical if the abnormalities of the insulin are subtle.

Increased proinsulin/insulin ratio in pancreas extracts of hyperglycemic rats.

The plasma ratio of proinsulin/insulin is raised in people with NIDDM. A relative hypersecretion of proinsulin is thought to be the cause, because pancreas extracts from diabetic rats have a raised proinsulin/insulin ratio. We tested the hypothesis that the pancreatic proinsulin/insulin mismatch results from hyperglycemia-induced beta-cell degranulation. Normal rats made hyperglycemic with 48-h glucose infusions had a raised pancreatic percentage of proinsulin. In contrast, rats infused with enough glucose to induce compensatory hyperinsulinemia without changing the plasma glucose level had a normal percentage of proinsulin. The raised percentage of proinsulin in the hyperglycemic rats reflected a reduction in pancreatic insulin content. Administering an inhibitor of insulin release, diazoxide, to hyperglycemic rats blocked the fall in pancreatic insulin content and prevented the rise in the percentage of proinsulin. Normal rats infused with tolbutamide for 3 days and enough glucose to maintain euglycemia had a 50% reduction in pancreatic insulin content. The beta-cell degranulation from this nonhyperglycemic mechanism resulted in a raised pancreatic percentage of proinsulin. In summary, chronic hyperglycemia causes beta-cell degranulation primarily because of hyperstimulated insulin release. The net result is a rise in the ratio of immature (proinsulin-rich) to mature (insulin-rich) granules, which is reflected as an increased relative proportion of proinsulin. Mobilization of these proinsulin-enriched granules may explain the relative hypersecretion of proinsulin that occurs with diabetes.

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.

Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm.

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.

Chromatographic heterogeneity of insulin extracted from insulomas.

On gel filtration of acid-ethanol extracts from three pancreatic beta-cell adenomas 1.4% to 1.8% of total immunomeasurable insulin (IMI) eluted ahead of proinsulin. This high molecular IMI was resolved into three components. The presence of urea in the dilute acetic acid solutions of extracted tumor tissue did not influence the pattern of gel filtration. High molecular IMI dissolved in dilute acetic acid showed to be stable if immediately rechromatographed, but a partial dissociation to insulinlike and proinsulinlike components (ILC and PLC) was found if rechromatography was performed after 48 h of incubation. Mainly ILC and PLC were found on rechromatography provided high molecular IMI was dissolved and incubated briefly in 0.04M phosphate buffer, pH 7.4. It proved improbable that the proteolytic action of some protein being extracted with the hormones caused a splitting of high molecular IMI at pH 7.4. We conclude from our findings that the components of high molecular IMI are not precursors of proinsulin and insulin but are either self-associated products of the hormones or associations of insulin and proinsulin to other proteins extracted from insuloma tissue.