Purification of porcine proinsulin by high-performance liquid chromatography

A procedure has been developed for purification of porcine proinsulin by high-performance liquid chromatography from a preparation obtained as a side product during the Sephadex G-50 gel filtration of an impure porcine insulin preparation. Reversed-phase chromatography was carried out on octadecylsilica as the stationary phase with graded mixtures of acetonitrile or methanol-acetonitrile and phosphate buffer pH 2.4 as the mobile phase. The crude preparation separated into five different groups of proteins, the proinsulin-containing peak being identified by the co-eluting internal proinsulin marker. After purification by conventional procedures (separation, pooling, freeze drying, desalting, reprecipitation and drying) this peak fraction was rechromatographed by high-performance liquid chromatography (for final purification) to give a single peak protein which had identical electrophoretic mobility to that of commercial porcine proinsulin, and which converted to a protein with electrophoretic mobility similar to that of porcine insulin.     

Semisynthesis of a shortened open-chain proinsulin

The openchain proinsulin model : B-chain-Arg-Arg-Ala-Gly-Lys-Arg-A-chain was prepared from partial protected native insulin chains and synthetic peptides.     

Separation and characterization of trypsin and carboxypeptidase B-digested products of Met-Lys-human proinsulin

Met-Lys-human proinsulin could be converted into insulin in vitro with the treatment of trypsin and carboxypeptidase B (CPB). Under less effective conditions, the enzymatic reaction does not proceed perfectly, and two main bands have been identified by native-polyacrylamide gel electrophoresis (PAGE) analysis. These two main products were thus separated and purified by DEAE-Sephadex A25 chromatography in a Tris-isopropanol system with an NaCl gradient. The isopropanol and NaCl were removed by a second DEAE-Sephadex column. Native-PAGE, mass spectrometric, and amino acid composition analyses indicate that one fraction of these two major products contains human insulin and desB30-insulin and that the other fraction is a mixture of human insulin analogs, which have one more basic amino acid than human insulin owing to the unsuitable amount of proteases, especially the lack of CPB. Furthermore, both receptor binding assay and radioimmunoassay have been utilized for the activity determination, and both fractions display almost full biological activity with porcine insulin as the standard. Present results provide further evidence for the quality control of recombinant human insulin production.     

Studies on human proinsulin C-peptide radioimmunoassay method--preparation of antiserum and its specificity-- (author's transl)]

The antisera using at final dilution of 1 : 10,000 have been prepared by immunizing synthetic human proinsulin connecting peptide to rabbits for human proinsulin C-peptide radioimmunoassay. The cross reactivities of human proinsulin C-peptide derivatives with the prepared antisera were reduced by leaving amino acid residues from N terminal, although this phenomenon was a little different among antisera. Those results suggested that main antigen determinant in N terminal 31-38 of human proinsulin connecting peptide. The cross reactivities of other animal proinsulin C-peptide and other peptide hormones with the prepared antissera were not recognized at 10(3) p mole/ml.     

Human proinsulin. C-peptide radioimmunoassay method. 125I labeling of human proinsulin. C-petide

125I-labelled human-C-peptide was prepared by chloramin T method, enzymic method and active ester method, respectively. Using respective 125I-labelled human-C-peptides in human proinsulin-C-peptide RIA, we compared the binding (Bo/T%) to antibody, displacement by standard human-C-peptide, the recovery test and stability. The usable 125I-labelled antigen for human proinsulin-C-peptide RIA could be prepared by chloramin T method and enzymic method wich labelled 125I to tyrosyl human proinsulin connecting peptide, and active ester method which conjugates 125I-labelled active ester to human proinsulin connecting peptide. The differences among those 125I-labelled antigens was not observed in displacement (B/Bo%) by standard human-C-peptide and the recovery test. In the case of constant preparation of 125I-labelled antigen for RIA, the enzymic method was the best from the viewpoint the reaction ratio is stable and stability of Bo/T% is good.     

Reversed-phase high-performance liquid chromatographic characterization of acetic acid extracts of the normal and the diabetic human pancreas

The combination of a divinylbenzene-based reversed-phase (RP) column and acetic acid gradients in water as mobile phase described in the accompanying paper was used for characterizing the extractable polypeptides from the normal and the diabetic human pancreas. The pancreas was lyophilized, minced and extracted three times in 3 M acetic acid. After mechanical clarification, the raw extracts were applied directly to the RP column. Alternatively, the extracts were lyophilized and subjected to size-exclusion chromatography on Sephadex G-50 in 3 M acetic acid. Two fractions with mol. wt. greater than 6000 dalton (Peak I) or with mol. wt. less than or equal to 6000 dalton (Peak II) were obtained. The Sephadex G-50 size-exclusion chromatography and the RP-high-performance liquid chromatographic (HPLC) analyses of the crude extracts from a normal pancreas clearly demonstrated the weight distribution and differences between the exocrine pancreas (containing primarily the major digestive enzymes) and the endocrine pancreas (containing insulin, glucagon, etc.). RP-HPLC analyses of crude extracts from various normal pancreatic glands resulted in very similar UV profiles, whereas those from a number of individual diabetic glands differed. Chromatograms of acetic acid extracts from normal pancreata were similar when analysed before or after lyophilization, whereas lyophilization of acetic acid extracts of diabetic glands resulted in severely obscured chromatograms. RP-HPLC analyses clearly demonstrated several differences between the diabetic and the normal pancreas. In the crude extracts, the extractable proteins from the diabetic pancreas were shifted towards lower molecular weight and/or hydrophobicity. Further, a peak co-eluting with authentic, human insulin could be demonstrated in the raw extract and in the peak II material from the normal pancreas, whereas virtually no mass signal was seen in the UV-profiles of similar materials from the diabetic gland. This finding was further verified by insulin radioimmunoassay (RIA) performed on the isolated fractions after RP-HPLC of a crude extract from a normal and a diabetic pancreas. The insulin content in the diabetic pancreas was found to be ca. 1% of that in the normal pancreas. When authentic glucagon was added to crude extracts from a diabetic pancreas, a single component was found after immediate analysis, but after several hours at room temperature the glucagon was found to be degraded. Added insulin was stable under these conditions. Similar RP analyses were performed on a silica C4 column eluted with an acetonitrile gradient in trifluoroacetic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

A High Performance Liquid Chromatographic Assessment of the Isolation of Bovine Proinsulin and a Synthetic Proinsulin Fragment

Abstract

High performance liquid chromatographic techniques have been used for the analysis and purification of the bovine proinsulin C-peptide fragment 34–45 (H-Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu-Leu-Ala-OH) (I) prepared solid phase synthetic methods. Conventional open column chromatographic methods failed to resolve the desired dodecapeptide (I)' from the des-Pro⁹-undecapeptide (II), which constituted the major solid phase synthetic deletion product. On 5- or 10- μm microparticulate reversed phase columns, these peptides are readily resolved preparatively in less than twenty minutes with simple elution systems. The elution order is in accord with that expected on the basis of hydrophobic fragmental constant summation. These HPLC techniques have been extended to permit the analytical assessment of the isolation of bovine pro-insulin and the proinsulin intermediates. The conversion of bovine proinsulin initially to the intermediates and finally to the desalanyl-insulin and the C-peptide on tryptic digestion can be followed by HPLC techniques which thus supplement alternative polyacrylamide disc electrophoretic methods.     

Preparation of Tyr-C-peptide from genetically altered human insulin precursor

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity.¹²⁵I labeled TyrC-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed inEscherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.     

A14-[125I]monoiodoinsulin purified by different high-performance liquid chromatographic procedures and by polyacrylamide gel electrophoresis: preparation, immunochemical properties and receptor binding affinity

Reversed-phase high-performance liquid chromatography (RP-HPLC) allows the rapid separation of A14-[125I]monoiodoinsulin directly from the iodination mixtures. It remains to be clarified, however, whether the RP-HPLC chromatographic conditions affect the properties of the purified tracer. In this study we prepared A14-[125I]insulin purified by polyacrylamide gel electrophoresis (PAGE) and by three different RP-HPLC mobile phases containing, respectively, ammonium acetate, sodium perchlorate and trifluoroacetic acid. The binding characteristics of all these tracers were examined using an insulin antiserum and insulin cell receptors. The specific radioactivity corresponded to the theoretical maximum for the RP-HPLC-purified tracers and was significantly lower for the PAGE-purified tracers. Significant differences were found in the binding of different tracers to the insulin antiserum: maximum binding ranged from 94 to 99% and was significantly lower for tracers purified by RP-HPLC eluents B and C; antiserum dilution giving 50% tracer binding was lower for tracers purified by RP-HPLC eluent B. The four insulin derivatives showed no difference in non-specific precipitation and in the affinity constant values calculated from the Scatchard analysis. No significant difference was found in the binding of the four insulin derivatives to the human-cultured IM-9 lymphocytes and to the human circulating monocytes. In conclusion, the present work demonstrates that the immunological properties of the A14-[125I]monoiodoinsulin purified by RP-HPLC may be partially affected by the composition of the mobile phase. In order to obtain a fully potent A14-[125I]insulin derivative and to have the possibility of comparing data from different laboratories, the chromatographic conditions must be taken into account.     

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.     

[B10Glu,B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B24和B25位之间插入Asp,构建了[B10Glu,B24-Asp-B25]胰岛素原基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性和凝胶过滤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经DEAE离子交换和RP-HPLC纯化得胰岛素突变体类似物.用凝胶过滤法测定了蛋白质分子自身的缔合性质,用圆二色谱测定了构象变化.放射性免疫活性及受体结合活性测定结果表明,突变体分子缔合性明显下降,放免活性和受体结合活性分别约为人胰岛素的73.6%和146%.     

Effect of two broad-spectrum antibiotics on activity and stability of continuous nitrifying system

A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristostatin, was introduced into an inactive human proinsulin molecule between the B29 and A2 sites to replace proinsulin C-peptide by molecular cloning techniques. The constructed Arg-Gly-Asp (RGD)-proinsulin gene was cloned into a temperature-inducible vector pBV220 and expressed in Escherichia coli. The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations. The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses. This RGD-proinsulin showed an inhibitory activity of adenosine 5′-diphosphate-induced human platelet aggregation with an IC₅₀ value of 200 nM. Its insulin receptor binding activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.     

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. Copyright © 2014 John Wiley & Sons, Ltd.     

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)₆ fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.     

Monitoring of recombinant human insulin production by narrow-bore reversed-phase high-performance liquid chromatography, high-performance capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry

An analytical scheme for monitoring recombinant human insulin (rhI) production is suggested. The scheme includes high-performance separation micro-techniques (narrow-bore RP-HPLC, HPCE) based on different separation mechanisms and matrix-assisted laser desorption ionisation time-of-flight MS, and allows one to obtain unambiguous information about purity and primary structure of all intermediates of the rhI production. The use of this scheme at all production steps provided optimisation of certain technological parameters [conditions for a fusion protein (FP) refolding, temperature and duration of the FP cleavage with trypsin, conditions for carboxypeptidase B digestion of di-ArgB31-B32-insulin] and achievement of a high purity of the end-product. The proposed scheme may be used for solving various problems in monitoring production of other recombinant proteins.     

Continuous mode of operation for large volume dosing in analytical carrier ampholyte-free isoelectric focusing of proteins applied to off-line detection of fractions

Mass spectrometry is being increasingly used for analysis of proteome complex samples. Sample preparation is often necessary to remove matrix interferences and to concentrate analytes prior to MS measurement. A useful method for this purpose is Carrier Ampholyte Free-Isoelectric Focusing (CAF-IEF). In this paper CAF-IEF of ampholytes was performed on a commercial apparatus EA101 (Villa Labeco, Slovakia) equipped with a specially made column for samples of large volume (up to 0.5 mL). A new continuous mode without voltage interruption or electrolyte replacement was developed. In this mode, a low molecular mass pI marker (PIM 7.4) and low concentrations of myoglobin and insulin (16 mg/L), respectively, were concentrated, and then 5-microL fractions collected for off-line analyses. The total time of focusing was 66 minutes. The concentration of PIM 7.4 in the fractions was increased up to 75 times (determined by UV-VIS spectrometry). The concentration in the fractions was increased up to 30 times for myoglobin and 10 times for insulin.     

The Removal of Zn(II) Through Calix[4]Recorcinarene Derivative Based Polymer Inclusion Membrane From Aqueous Solution

In the present work, the transport of Zn(II) metal ion from an aqueous nitrate solution of different metal ions through a polymer inclusion membrane (PIM) containing calix[4]resorcinarene derivative used as a carrier were investigated. Zn(II)metal ion showing high permeability were transported through PIMs prepared from cellulose triacetate (CTA) as a polymeric support material and 2-NPOE as a plasticizer. Total Zn(II) concentration was determined with an Atomic Absorption Spectrometer (AAS) in the acceptor phase. The prepared PIM and supported liquid membrane (SLM) were characterized by using Scanning Electron Microscopy(SEM) and Atomic Force Microscopy (AFM) techniques. The effects of membrane composition, effects of type of plasticizer in the membrane, effects of carrier concentration, and the thickness of the membranes were examined in the facilitated transport experiments of Zn(II) ion through PIM. We compared the performance of SLM experiments under the optimum conditions identified by the PIM studies. Higher permeability coefficient values for Zn(II) was found for SLM, while lower values were ascertained for PIM. The kinetic parameters which have been calculated as the constant rate (k), permeability coefficient (P), flux (J) and diffusion coefficient (D).     

Micro sequential injection: automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.     

Ammonia-recycled percolation process for pretreatment of biomass feedstock

The construction of a gene encoding Lys-human proinsulin, its direct expression inE. coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20–30%. After simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. After separation with DEAE-Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L of fermentation medium.     

Studies on the Renaturation with Simultaneous Purification of Recombinant Human Proinsulin with Unit of Simultaneous Renaturation and Purification of Protein in Semi－preparative Scale

The recombinant human proinsulin (rh-proinsulin) is the precursor of insulin, which is connected with C- peptide between the carboxyl-terminal of chain A and NH2-terminal of chain B of insulin. In the presences of trypsin and carboxypeptides B (CPB), the C-peptide can be cleaved from the special two peptides of rh-proinsulin and the recombinant human insulin (rh-insulin) can be, thus, obtained1,2. The rh-proinsulin expressed in E. coli exists as inclusion body, hardly dissolves in water,…