Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form

(MTG) The Streptoverticillium transglutaminase gene, synthesized previously for yeast expression, was modified and resynthesized for overexpression in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. coli transformed with pUCTRPMTG02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200-300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli.     

A two-step refolding of acid-denatured microbial transglutaminase escaping from the aggregation-prone intermediate

Microbial transglutaminase (MTG) is a monomeric globular enzyme made of 331 amino acid residues. The conformation of MTG was examined over the pH 2.0-6.0 region using circular dichroism (CD) and 1-anilino-8-naphthalenesulfonate (ANS). Under conditions of low ionic strength, a decrease of pH below 4 caused a stepwise unfolding with an intermediate exhibiting specific ANS-binding before full unfolding at pH 2.0. At high ionic strength, the decrease of pH led to only an intermediate without further unfolding. The intermediate corresponds to the molten globule state with a secondary structure similar to the native state but disordered tertiary structures. A pH- and NaCl concentration-dependent phase diagram showed that the fully unfolded state exists only under limited conditions of low pH and a low NaCl concentration. Although a refolding yield by the direct jump to pH 6.0 was low, a two-step refolding with incubation at pH 4.0, where MTG is marginally stable, and a subsequent jump to pH 6.0 improved the yield by suppressing the kinetic traps. We propose that the two-step refolding is useful for improving the yield of larger proteins with a high pI value.     

In vitro refolding of PEGylated lipase

Covalent modification of proteins with polyethylene glycol (PEG) has become a well established drug enhancement strategy in the biopharmaceutical industry. The general benefits of PEGylation, such as prolonged serum half-lives or reduced in vivo immunogenicity, are well known. To date, the PEGylation process has been performed with purified proteins, which often requires additional multi-step purification steps to harvest the desired PEGylate. However, it would be beneficial for bioprocessing if 'renaturation,' i.e. in vitro refolding and 'modification,' and PEGylation can be integrated, especially for inclusion body proteins. We investigated the feasibility of protein PEGylation under denaturing conditions and of protein refolding with the attached PEG molecule. Using lipase as a model protein, PEGylation occurred in 8 M urea and covalently attached PEG did not appear to hinder subsequent refolding.     

In vitro protein refolding by chromatographic procedures

In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important polypeptides that are overexpressed in Escherichia coli. This review focuses on protein refolding methods based on column procedures because recent advances in chromatographic refolding have shown promising results.     

Stable intermediates can be trapped during the reversible refolding of urea-denatured rhodanese

The enzyme rhodanese (EC 2.8.1.1) could be reversibly refolded from urea in the presence of lauryl maltoside, beta-mercaptoethanol, and sodium thiosulfate. The unfolding/folding transition monitored using intrinsic fluorescence was resolved into two two-state transitions with midpoints at 3.6 and 5.0 M urea. The analysis assumed an intermediate with an emission maximum at 345 nm. Monitoring anisotropy of intrinsic fluorescence also gave an asymmetric transition. Activity followed one two-state transition centered at 3.6 M urea with no major change of secondary structure. Without thiosulfate or mercaptoethanol, there was one two-state transition at 5.0 M urea giving a species, in dilute urea, with a fluorescence maximum at 345 nm. This intermediate slowly relaxed toward 335 nm (t1/2 = 85 min) if only thiosulfate was absent but without regaining activity. Subsequent addition of thiosulfate led to a first-order recovery of activity (t1/2 = 75 min). Thus, a possible folding intermediate can be trapped which displays increased access of water and solutes to its fluorescent tryptophans. This intermediate conformer, which is flexible, has considerable secondary structure, is inactive, has exposed hydrophobic surfaces, and requires specific reducing conditions to regain full activity. Refolding probably involves an initial, rapid, hydrophobic collapse with acquisition of secondary structure to form the intermediate, followed by slower adjustment to the native global conformation. Final reactivation requires reduction localized at the active site.     

Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli

In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M _r∼55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca²⁺- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I₁ (existing in 1.8–1.4 M urea) and I₂ (existing in 0.8–0.4 M urea). I₁ was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I₂ was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates (I_B). Comparison of the properties of these intermediates suggested that I_B in the kinetic process corresponded to I₁ in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.     

Enhancement of Streptomyces transglutaminase activity and pro-peptide cleavage efficiency by introducing linker peptide in the C-terminus of the pro-peptide

Streptomyces transglutaminase (TGase) has been widely used in food, pharmaceutical and textile industries. Streptomyces TGase is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by removing its N-terminal pro-peptide. Although the pro-peptide is essential for TGase folding and secretion, few studies have been reported on improving the properties of TGase by pro-peptide engineering. In this study, we developed a new approach to improve the properties of TGase based on pro-peptide engineering. When the α-helix^37G?42S in pro-peptide was substituted with three glycines and three alanines respectively, the mutants exhibited higher specific activity and the efficiency of pro-peptide cleavage was enhanced. To further improve the properties of TGase, relevant mutations were constructed by introducing linker peptides in the C-terminus of the pro-peptide. Mutants with GS (GGGGS) and PT (PTPPTTPT) linker peptide exhibited 1.28 fold and 1.5 fold higher specific activity than the wild-type enzyme, respectively. This new method could be used to improve the properties of TGase by pro-peptide modification, which is a promising technology for creating unique TGase with various beneficial properties.     

Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase

The course of the recovery of the enzymatic activity and the native conformation during the renaturation of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. Under suitable conditions, an activity recovery of 95% can be obtained and the reactivation follows a triphasic course. The initial two phases are relatively fast, whereas the slow phase takes some 24 h to reach completion. The recovery of the native conformation has been followed by changes in fluorescence, ultraviolet absorption and in exposed SH groups and has been shown to be a biphasic process. Both the reactivation and the refolding processes are independent of protein concentrations within a certain range, showing that the dimerization of the enzyme molecule is not rate-limiting. A comparison of the rate constants for the refolding of the molecule with those for the recovery of its catalytic activity shows that these are not synchronized and the activity recovery approaches completion after the refolding and dimerization of the subunits so far as can be detected by the methods employed. The final stage of refolding with complete activity recovery probably involves subtle conformational changes of the dimeric enzyme molecule not detectable by the physiochemical methods used in the present study.     

The refolding of urea-denatured ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase

The refolding of urea-denatured ribonuclease A was measured at 0.31-3.1 mol . l-1 urea in the presence of various concentrations of peptidyl-prolyl cis-trans isomerase isolated from pig kidney. The rate of the slow CT-phase in the refolding reaction was found to be sensitive to this enzyme. A rate enhancement proportional to the isomerase activity has been observed. The activity of the enzyme was assayed with Glt-Ala-Ala-Pro-Phe-4-nitroanilide. The catalytic activity of the isomerase against unfolded ribonuclease is suppressed after preincubation of the enzyme with 0.001 mol . l-1 Cu2+, 0.01 mol . l-1 H+ and by heat inactivation. The results indicate the involvement of the cis/trans interconversion of proline peptide bonds during the refolding of ribonuclease A.     

Effect of Zn2+ during reactivation and refolding of urea-denatured creatine kinase

The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotrans-ferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn²⁺ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the molten globule state in the CK folding pathway. During refolding in the presence of Zn²⁺ , creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.     

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.     

Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.     

In vitro refolding of porcine pepsin immobilized on agarose beads

Since in vitro refolding of pepsin has long been attempted without success, it has been suspected that pepsin has no intrinsic refolding ability. In the present study, in order to eliminate unfavorable intermolecular interactions bringing about aggregation and autoproteolysis, we immobilized pepsin onto agarose beads. This technique enabled us to search extensively for appropriate refolding conditions without limitation of the refolding period. Renaturation of immobilized pepsin was observed exclusively at pH 3-5. This process was extremely slow and reached equilibrium after 300 h. Sixty percent of the proteolytic activity was recovered at pH 5. Addition of salts raised the recovery to 80% but had no significant effect on the refolding rate, suggesting that the salts mainly stabilize the native state of pepsin. This is the first report on the successful in vitro refolding of pepsin.     

Altered secretary efficiency of Streptomyces hygroscopicus transglutaminase in Escherichia coli by the pro-peptide modification

Streptomyces transglutaminase (TGase) is naturally synthesized as a zymogen (pro-TGase), which is then processed to produce the active enzyme through removal of its pro-peptide. In this study, we investigated the effect of the pro-peptide on the secretion of Streptomyces hygroscopicus TGase in Escherichia coli by modifying its pro-peptide. Four N-terminal amino acid residues (Tyr12, Asn27, Asn30, and Arg32) in the pro-peptide predicted to interact with TGase region through hydrogen bonds. When the four amino acid residues were mutated into Ala, the secretion of TGase was partially or completely inhibited. Furthermore, deletion of the C-terminal α-helix^37–42 in the pro-peptide concomitantly decreased the secretion of TGase. However, deletion of the C-terminal loop^43–52 of the pro-peptide, resulted in increased secretion of TGase by approximately 70% as compared with the control native pro-peptide. These findings indicate that modification of the pro-peptide has a significant impact on the secretory efficiency of TGase in E. coli.     

Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase

Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified. In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase. Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution. Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified. The optimized refolding procedure is reported.     

Affinity precipitation and macroaffinity ligand facilitated three-phase partitioning for refolding and simultaneous purification of urea-denatured pectinase

Protein refolding is an integral step in the recovery of protein activity from inclusion bodies. It is shown that affinity precipitation and macroaffinity ligand facilitated three-phase partitioning (MLFTPP) led to refolding of urea-denatured pectinase present in a commercial preparation, with simultaneous purification. Affinity precipitation consists of precipitation of the desired enzyme by complexing it with a suitable stimulus-sensitive macroaffinity ligand. This ligand in this case was alginate/esterified alginate. The complex of the polymer-pectinase could be precipitated by adding calcium ions. In MLFTPP (carried out by adding tertiary butanol and ammonium sulfate to the aqueous solution of crude enzyme and the polymer), the polymer or its complex with the enzyme form an interfacial precipitate between tert-butyl alcohol phase and aqueous phase. It is believed that in both processes, while molecular recognition of alginate/esterified alginate to pectinase facilitates their selective binding to the enzyme, the correct refolding is facilitated by preventing molecular aggregation of unfolded enzyme molecules. Three-phase partitioning with esterified alginate as the macroaffinity ligand gave 100% recovery with 4-fold purification. Affinity precipitation with 1% alginate gave 52% yield with 18-fold purification. On the other hand, use of 0.5% esterified alginate gave only 7-fold purification but with 75% recovery of activity.     

Properties and applications of microbial transglutaminase

Some properties and applications of the transglutaminase (TGase) referred to as microbial TGase (MTGase), derived from a variant of Streptomyces mobaraensis (formerly classified as Streptoverticillium mobaraense), are described. MTGase cross-linked most food proteins, such as caseins, soybean globulins, gluten, actin, myosins, and egg proteins, as efficiently as mammalian TGases by forming an -(-glutamyl)lysine bond. However, unlike many other TGases, MTGase is calcium-independent and has a relatively low molecular weight. Both of these properties are of advantage in industrial applications; a number of studies have illustrated the potential of MTGase in food processing and other areas. The crystal structure of MTGase has been solved. It provides basic structural information on the MTGase and accounts well for its characteristics. Moreover, an efficient method for producing extracellular MTGase has been established using Corynebacterium glutamicum. MTGase may be expected to find many uses in both food and non-food applications.     

In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis

Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.     

微生物转谷氨酰胺酶的纯化方法和酶学性质研究

由Streptoverticillium mobaTaense发酵生产的转谷氨酰胺酶经过除茵体、超滤浓缩、乙醇沉淀、干燥后得到粗酶产品,其活力回收率约70％。又经Superdex-75凝胶过滤和Source 30S阳离子交换两步纯化后得到纯酶,最终酶活力收率约37％。酶最适温度为50℃,在40℃以下稳定性良好;最适pH为6．0,pH4．0～8．0时比较稳定。离子强度对酶影响很小。