Zhan-Yun Guo | Tongji University (original) (raw)

Papers by Zhan-Yun Guo

Research paper thumbnail of The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1

Science in China Series C, 2002

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homo... more Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermodynamic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a single-chain hybrid of insulin and IGF-1, [B10Glu]Ins/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides isomer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.

Research paper thumbnail of Peptide models of four possible insulin folding intermediates with two disulfides

Protein Science, 2009

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kin... more The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.

Research paper thumbnail of A peptide model of insulin folding intermediate with one disulfide

Protein Science, 2003

Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our ... more Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.

Research paper thumbnail of Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

Protein Engineering Design and Selection, 2000

the effects of different residues on insulin biosynthesis and The residues A21Asn, B12Val, B16Tyr... more the effects of different residues on insulin biosynthesis and The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, folding. They found that A19Tyr, B11Leu and B13Glu are B26Tyr and B27Thr, buried in the dimer of insulin, were essential for receptor binding, especially the A19 residue, and identified by means of alanine-scanning mutagenesis. The they proposed an insulin receptor binding site which consists receptor binding activity, in vivo biological potency and of B6Leu, B8Gly, B23Gly, B24Phe, A2Ile, A3Val and A19Tyr. self-association properties of the seven single alanine human However, they only obtained seven purified analogs and did insulin mutants were determined. Four of the seven single not measure their association properties, although this is alanine mutants, [B12Ala]human insulin, [B16Ala]human important for the clinical use of insulin. insulin, [B24Ala]human insulin and [B26Ala]human insu-To elucidate the role of the residues buried in the dimer on lin, are monomeric insulin, which indicates that B12Val, the association properties of insulin and to screen some B16Tyr, B24Phe and B26Tyr are crucial for the formation monomeric insulins, we mutated the seven residues by alanineof insulin dimer. The monomeric [B16Ala]human insulin scanning mutagenesis. Here we report four new monomeric and [B26Ala]human insulin retain 27 and 54% receptor insulins. Two of them, [B16Ala]human insulin and [B26Ala]binding activity, respectively, and nearly the same in vivo human insulin, retain high in vitro and in vivo biological biological potency compared with native insulin, so they activities and could be developed as the fast-acting insulin could be developed as the fast-acting insulin. required in clinical practice. Keywords: alanine-scanning mutagenesis/dimer-forming surface/insulin/monomer Materials and methods Microbial strains, plasmids and cloning procedures The Escherichia coli strains used were TG1 and RZ1032

Research paper thumbnail of In Vitro Folding/Unfolding of Insulin/Single-Chain Insulin

Protein & Peptide Letters, 2006

Insulin is a double-chain (designated A and B chain respectively) protein hormone containing thre... more Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.

Research paper thumbnail of Equilibrium Folding of Porcine Insulin Precursor in the Presence of Redox Buffer: Implications for the Common Intermediates Shared by Its Unfolding/ Refolding Processes

Protein & Peptide Letters, 2008

We use the procedure established for &amp... more 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.

Research paper thumbnail of The Active Site His-460 of Human Acyl-coenzyme A:Cholesterol Acyltransferase 1 Resides in a Hitherto Undisclosed Transmembrane Domain

Journal of Biological Chemistry, 2005

Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endopl... more Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endoplasmic reticulum (ER). We had previously reported that hACAT1 contains seven transmembrane domains (TMD) (

Research paper thumbnail of Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin

Biological Chemistry, 2004

Insulin contains three disulfide bonds, one intrachain bond, A6–A11, and two interchain bonds, A7... more Insulin contains three disulfide bonds, one intrachain bond, A6–A11, and two interchain bonds, A7–B7 and A20–B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7–B7 were replaced by serine) showed that disulfide A7–B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7–B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant...

Research paper thumbnail of Putative Disulfide-Forming Pathway of Porcine Insulin Precursor during Its Refolding in Vitro †

Biochemistry, 2001

Although the structure of insulin has been well studied, the formation pathway of the three disul... more Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.

Research paper thumbnail of The Disulfide Linkage and the Free Sulfhydryl Accessibility of Acyl-Coenzyme A:Cholesterol Acyltransferase 1 As Studied by Using mPEG 5000 -Maleimide †

Biochemistry, 2005

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasm... more Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiolspecific modifications by mPEG 5000-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.

Research paper thumbnail of Functionality of the Seventh and Eighth Transmembrane Domains of Acyl-Coenzyme A:Cholesterol Acyltransferase 1 †

Biochemistry, 2007

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic ret... more Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic reticulum. ACAT1 is a homotetrameric protein and contains nine transmembrane domains (TMDs). His460 is a key active residue and is located within TMD7. Human ACAT1 has seven free Cys, but the recombinant ACAT1 devoid of free Cys retains full enzyme activity. To further probe the functionality of TMD7 (amino acids 446-460) and TMD8 (amino acids 466-481), we used a parental ACAT1 devoid of free Cys as the template to perform Cys-scanning mutagenesis within these regions. Each of the single Cys mutants was expressed in Chinese hamster ovary (CHO) cell line AC29 lacking endogenous ACAT1. We measured the effect of single Cys substitution on enzyme activity and used the Cu(1,10-phenanthroline) 2 SO 4-mediated disulfide cross-linking method to probe possible interactions of engineered Cys between the two identical subunits. The results show that several residues in one subunit closely interact with the same residues in the other subunit; mutating these residues to Cys does not lead to large loss in enzyme activity. Helical wheel analysis suggests that these residues are located at one side of the coil. In contrast, mutating residues F453, A457, or H460 to Cys causes large loss in enzyme activity; the latter residues are located at the opposite side of the coil. A similar arrangement is found for residues in TMD8. Thus, helical coils in TMD7 and TMD8 have two distinct functional sides: one side is involved in substrate-binding/catalysis, while the other side is involved in subunit interaction.

Research paper thumbnail of Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a

Biochemical Journal, 2020

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagoni... more Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin–GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved...

Research paper thumbnail of Development of a selective agonist for relaxin family peptide receptor 3

Scientific reports, Jan 12, 2017

Relaxin family peptides perform a variety of biological functions by activating four G protein-co... more Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23-B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [...

Research paper thumbnail of Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Scientific reports, Jun 12, 2017

Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. ... more Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker's highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate th...

Research paper thumbnail of Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4

Scientific reports, Jul 11, 2016

The relaxin family peptides play a variety of biological functions by activating four G protein-c... more The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1-4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr, and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-c...

Research paper thumbnail of Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family

PloS one, 2016

We recently developed novel bioluminescent binding assays for several protein/peptide hormones to... more We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s w...

Research paper thumbnail of Acyl-coenzyme A: cholesterol acyltransferase family

Frontiers of Biology in China, 2008

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for... more The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer's disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.

Research paper thumbnail of Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris

Amino acids, Jan 5, 2015

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Matu... more Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one lit...

Research paper thumbnail of The electrostatic interactions of relaxin-3 with receptor RXFP4 and the influence of its B-chain C-terminal conformation

The FEBS journal, 2014

Relaxin-3 (also known as insulin-like peptide 7) is an insulin/relaxin-superfamily peptide hormon... more Relaxin-3 (also known as insulin-like peptide 7) is an insulin/relaxin-superfamily peptide hormone that can bind and activate three relaxin-family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin-3 and its cognate receptor RXFP3 by using a charge-exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin-3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100-104) motif that is essential for RXFP4 activation by relaxin-3. Mutagenesis of the conserved positively charged Arg residues of relaxin-3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B2...

Research paper thumbnail of Recombinant expression, different downstream processing of the disulfide-rich anti-tumor peptide Ranpirnase and its effect on the growth of human glioma cell line SHG-44

Biomedical reports, 2013

Ranpirnase (Onconase) is a frogspawn-derived disulfide-rich peptide with ribonuclease activity th... more Ranpirnase (Onconase) is a frogspawn-derived disulfide-rich peptide with ribonuclease activity that may be used for tumor treatment. In the present study, we established an efficient approach for preparing mature ranpirnase which may be used for research and therapeutic purposes. The designed ranpirnase precursors carried a 6xHis-tag and were recombinantly expressed in Escherichia coli. After S-sulfonation, the precursors were purified by immobilized metal-ion affinity chromatography. Following removal of the tag by aminopeptidase cleavage, cyclization and in vitro oxidative refolding, the mature ranpirnase was obtained with considerable yield, and the yield of mature ranpirnase was ~50-60 mg per liter cultures. In addition, ranpirnase inhibited the growth of human glioma cells SHG-44 in a dose-dependent manner. Thus the present study has provided an efficient approach for the preparation of active ranpirnase and its analogues for future studies.

Research paper thumbnail of The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1

Science in China Series C, 2002

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homo... more Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermodynamic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a single-chain hybrid of insulin and IGF-1, [B10Glu]Ins/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides isomer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.

Research paper thumbnail of Peptide models of four possible insulin folding intermediates with two disulfides

Protein Science, 2009

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kin... more The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.

Research paper thumbnail of A peptide model of insulin folding intermediate with one disulfide

Protein Science, 2003

Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our ... more Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.

Research paper thumbnail of Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

Protein Engineering Design and Selection, 2000

the effects of different residues on insulin biosynthesis and The residues A21Asn, B12Val, B16Tyr... more the effects of different residues on insulin biosynthesis and The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, folding. They found that A19Tyr, B11Leu and B13Glu are B26Tyr and B27Thr, buried in the dimer of insulin, were essential for receptor binding, especially the A19 residue, and identified by means of alanine-scanning mutagenesis. The they proposed an insulin receptor binding site which consists receptor binding activity, in vivo biological potency and of B6Leu, B8Gly, B23Gly, B24Phe, A2Ile, A3Val and A19Tyr. self-association properties of the seven single alanine human However, they only obtained seven purified analogs and did insulin mutants were determined. Four of the seven single not measure their association properties, although this is alanine mutants, [B12Ala]human insulin, [B16Ala]human important for the clinical use of insulin. insulin, [B24Ala]human insulin and [B26Ala]human insu-To elucidate the role of the residues buried in the dimer on lin, are monomeric insulin, which indicates that B12Val, the association properties of insulin and to screen some B16Tyr, B24Phe and B26Tyr are crucial for the formation monomeric insulins, we mutated the seven residues by alanineof insulin dimer. The monomeric [B16Ala]human insulin scanning mutagenesis. Here we report four new monomeric and [B26Ala]human insulin retain 27 and 54% receptor insulins. Two of them, [B16Ala]human insulin and [B26Ala]binding activity, respectively, and nearly the same in vivo human insulin, retain high in vitro and in vivo biological biological potency compared with native insulin, so they activities and could be developed as the fast-acting insulin could be developed as the fast-acting insulin. required in clinical practice. Keywords: alanine-scanning mutagenesis/dimer-forming surface/insulin/monomer Materials and methods Microbial strains, plasmids and cloning procedures The Escherichia coli strains used were TG1 and RZ1032

Research paper thumbnail of In Vitro Folding/Unfolding of Insulin/Single-Chain Insulin

Protein & Peptide Letters, 2006

Insulin is a double-chain (designated A and B chain respectively) protein hormone containing thre... more Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.

Research paper thumbnail of Equilibrium Folding of Porcine Insulin Precursor in the Presence of Redox Buffer: Implications for the Common Intermediates Shared by Its Unfolding/ Refolding Processes

Protein & Peptide Letters, 2008

We use the procedure established for &amp... more 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.

Research paper thumbnail of The Active Site His-460 of Human Acyl-coenzyme A:Cholesterol Acyltransferase 1 Resides in a Hitherto Undisclosed Transmembrane Domain

Journal of Biological Chemistry, 2005

Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endopl... more Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endoplasmic reticulum (ER). We had previously reported that hACAT1 contains seven transmembrane domains (TMD) (

Research paper thumbnail of Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin

Biological Chemistry, 2004

Insulin contains three disulfide bonds, one intrachain bond, A6–A11, and two interchain bonds, A7... more Insulin contains three disulfide bonds, one intrachain bond, A6–A11, and two interchain bonds, A7–B7 and A20–B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7–B7 were replaced by serine) showed that disulfide A7–B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7–B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant...

Research paper thumbnail of Putative Disulfide-Forming Pathway of Porcine Insulin Precursor during Its Refolding in Vitro †

Biochemistry, 2001

Although the structure of insulin has been well studied, the formation pathway of the three disul... more Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.

Research paper thumbnail of The Disulfide Linkage and the Free Sulfhydryl Accessibility of Acyl-Coenzyme A:Cholesterol Acyltransferase 1 As Studied by Using mPEG 5000 -Maleimide †

Biochemistry, 2005

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasm... more Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiolspecific modifications by mPEG 5000-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.

Research paper thumbnail of Functionality of the Seventh and Eighth Transmembrane Domains of Acyl-Coenzyme A:Cholesterol Acyltransferase 1 †

Biochemistry, 2007

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic ret... more Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic reticulum. ACAT1 is a homotetrameric protein and contains nine transmembrane domains (TMDs). His460 is a key active residue and is located within TMD7. Human ACAT1 has seven free Cys, but the recombinant ACAT1 devoid of free Cys retains full enzyme activity. To further probe the functionality of TMD7 (amino acids 446-460) and TMD8 (amino acids 466-481), we used a parental ACAT1 devoid of free Cys as the template to perform Cys-scanning mutagenesis within these regions. Each of the single Cys mutants was expressed in Chinese hamster ovary (CHO) cell line AC29 lacking endogenous ACAT1. We measured the effect of single Cys substitution on enzyme activity and used the Cu(1,10-phenanthroline) 2 SO 4-mediated disulfide cross-linking method to probe possible interactions of engineered Cys between the two identical subunits. The results show that several residues in one subunit closely interact with the same residues in the other subunit; mutating these residues to Cys does not lead to large loss in enzyme activity. Helical wheel analysis suggests that these residues are located at one side of the coil. In contrast, mutating residues F453, A457, or H460 to Cys causes large loss in enzyme activity; the latter residues are located at the opposite side of the coil. A similar arrangement is found for residues in TMD8. Thus, helical coils in TMD7 and TMD8 have two distinct functional sides: one side is involved in substrate-binding/catalysis, while the other side is involved in subunit interaction.

Research paper thumbnail of Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a

Biochemical Journal, 2020

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagoni... more Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin–GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved...

Research paper thumbnail of Development of a selective agonist for relaxin family peptide receptor 3

Scientific reports, Jan 12, 2017

Relaxin family peptides perform a variety of biological functions by activating four G protein-co... more Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23-B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [...

Research paper thumbnail of Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Scientific reports, Jun 12, 2017

Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. ... more Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker's highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate th...

Research paper thumbnail of Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4

Scientific reports, Jul 11, 2016

The relaxin family peptides play a variety of biological functions by activating four G protein-c... more The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1-4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr, and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-c...

Research paper thumbnail of Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family

PloS one, 2016

We recently developed novel bioluminescent binding assays for several protein/peptide hormones to... more We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s w...

Research paper thumbnail of Acyl-coenzyme A: cholesterol acyltransferase family

Frontiers of Biology in China, 2008

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for... more The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer's disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.

Research paper thumbnail of Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris

Amino acids, Jan 5, 2015

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Matu... more Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one lit...

Research paper thumbnail of The electrostatic interactions of relaxin-3 with receptor RXFP4 and the influence of its B-chain C-terminal conformation

The FEBS journal, 2014

Relaxin-3 (also known as insulin-like peptide 7) is an insulin/relaxin-superfamily peptide hormon... more Relaxin-3 (also known as insulin-like peptide 7) is an insulin/relaxin-superfamily peptide hormone that can bind and activate three relaxin-family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin-3 and its cognate receptor RXFP3 by using a charge-exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin-3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100-104) motif that is essential for RXFP4 activation by relaxin-3. Mutagenesis of the conserved positively charged Arg residues of relaxin-3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B2...

Research paper thumbnail of Recombinant expression, different downstream processing of the disulfide-rich anti-tumor peptide Ranpirnase and its effect on the growth of human glioma cell line SHG-44

Biomedical reports, 2013

Ranpirnase (Onconase) is a frogspawn-derived disulfide-rich peptide with ribonuclease activity th... more Ranpirnase (Onconase) is a frogspawn-derived disulfide-rich peptide with ribonuclease activity that may be used for tumor treatment. In the present study, we established an efficient approach for preparing mature ranpirnase which may be used for research and therapeutic purposes. The designed ranpirnase precursors carried a 6xHis-tag and were recombinantly expressed in Escherichia coli. After S-sulfonation, the precursors were purified by immobilized metal-ion affinity chromatography. Following removal of the tag by aminopeptidase cleavage, cyclization and in vitro oxidative refolding, the mature ranpirnase was obtained with considerable yield, and the yield of mature ranpirnase was ~50-60 mg per liter cultures. In addition, ranpirnase inhibited the growth of human glioma cells SHG-44 in a dose-dependent manner. Thus the present study has provided an efficient approach for the preparation of active ranpirnase and its analogues for future studies.