Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.     

利用内蛋白子剪切功能一步纯化重组人神经营养因子-3

将人神经营养因子 - 3(h NT3)基因插入含内蛋白子 -几丁质结合区 (Intein- CBD)片段的质粒p TXB1的多克隆位点 ,构建成重组子 p TXB- h NT3,随后转化入 E.coli 2 566并进行融合表达 .表达产物包涵体经 8mol/ L脲变性 ,并在 GSH,GSSG存在下复性 .复性后的融合蛋白经几丁质珠亲和柱吸附 .待洗涤杂蛋白后 ,加入 50 mmol/ L DTT在 4℃或 2 5℃进行剪切反应 48h,再用缓冲液洗脱 ,即得 h NT3.SDS- PAGE分析表明 ,h NT3达电泳纯 .其分子量约为 1 4 k D     

In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions

Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.     

重组环状胸腺五肽结构类似物Cyclo-（Cys- Arg-Lys –Asp-Val-Tyr）的制备、鉴定和活性检测

为了实现蛋白内含肽(Intein)介导的重组环状胸腺五肽结构类似物［cyclo-（Cys -Arg-Lys –Asp-Val-Tyr）,cTP］的高效制备,设计并合成编码6个氨基酸的cTP基因,克隆到表达载体pTWIN1,重组表达质粒pTW-cTp转化E.coli ER2566构建工程菌,IPTG诱导由几丁质结合域纯化标签（chitin binding domain,CBD）、2个蛋白内含肽和目的多肽组成的“多元”融合蛋白(CBD-intein1-cTP-intein2-CBD)的高效表达.几丁质柱亲合层析纯化融合蛋白后,改变pH值和温度诱导intein1 C端切割,硫醇MESNA诱导intein2 N端切割,释放N端为Cys,C端为硫酯的重组cTP线性前体,通过非保护多肽硫酯环合法实现环肽生成.激光飞行质谱结果显示,纯化产物的分子量为764.4,与环肽的理论值相符.免疫活性检测结果显示,环肽cTP较线性多肽TP-5具有更显著的促进巨噬细胞吞噬能力的活性（P<0.01）和促进B细胞抗体生成的活性（P<0.01）.     

Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening

Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.     

Cleavage and purification of intein fusion proteins using the Streptococcus gordonii spex system

A gram-positive bacterial expression vector using Streptococcus gordonii has been developed for expression and secretion, or surface anchoring of heterologous proteins. This system, termed Surface Protein Expression system or SPEX, has been used to express a variety of surface anchored and secreted proteins. In this study, the Mycobacterium xenopi (Mxe) GyrA intein and chitin binding domain from Bacillus circulans chitinase Al were used in conjunction with SPEX to express a fusion protein to facilitate secretion, cleavage, and purification. Streptococcus gordonii was transformed to express a secreted fusion protein consisting of a target protein with a C-terminal intein and chitin-binding domain. Two target proteins, the C-repeat region of the Streptococcus pyogenes M6 protein (M6) and the nuclease A (NucA) enzyme of Staphylococcus aureus, were expressed and tested for intein cleavage. The secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cleavage to release the target protein. The M6 and NucA fusion proteins were shown to bind chitin beads and elute under cleavage reaction conditions. In addition, NucA demonstrated enzyme activity both before and after intein cleavage.     

Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein

Advances in expressed protein ligation (EPL) methods that permit specific introduction of unique modifications into proteins have facilitated protein engineering, structure-function and protein interaction studies. An EPL-generated hybrid exchangeable apolipoprotein has been constructed from recombinant fragments of apolipoprotein E (apoE) and apolipophorin III (apoLp-III). A recombinant fusion protein comprised of human apoE N-terminal residues 1-111, a modified Saccharomyces cerevisiae intein and a chitin binding domain was subjected to 2-mercaptoethanesulfonic acid (MESNA) induced cleavage to generate apoE(1-111)-MESNA. A second fusion protein was comprised of a bacterial pelB leader peptide fused to a variant form of Galleria mellonella apoLp-III residues 1-91. The N-terminal pelB leader sequence directed the newly synthesized fusion protein to the Escherichia coli perisplamic space where endogenous leader peptidase cleavage generated the desired N-terminal cysteine-containing protein fragment. The resulting apoLp-III fragment, which contained no sequence tags or tails, escaped the bacteria and accumulated in the culture medium. When cultured in M9 minimal medium, Asp1Cys apoLp-III(1-91) was produced in high yield and was the sole major protein in the culture supernatant. Ligation reactions with apoE(1-111)-MESNA yielded an engineered hybrid apolipoprotein. The results document the utility of the pelB fusion protein system for generating active N-terminal cysteine containing proteins for EPL applications.     

A pH‐induced,intein‐mediated expression and purification of recombinant human epidermal growth factor in Escherichia coli

Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH‐induced intein and a chitin‐binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitin‐binding domain fusion‐tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH‐induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self‐cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion‐exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat‐resistant and acid‐resistant recombinant proteins. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:758–764, 2015     

重组血管活性肠肽的制备及鉴定

为利用基因工程技术获得重组血管活性肠肽(vasoactive intestinal peptide,VIP),根据大肠杆菌的密码偏好性,设计并人工合成编码28个氨基酸的VIP基因。克隆到表达载体PTWIN,构建重组质粒PTWIN-VIP,转化宿主菌E. coli Strain ER2566,构建表达工程菌。实现由重组VIP,内含肽与纤维素结合域（cellulose binding domain, CBD）组成的融合蛋白表达。融合蛋白经几丁质亲和层析纯化,通过改变温度和缓冲液PH值切割融合蛋白,获得目的多肽。所得的多肽经质谱测定分子量结果与理论值相符。生物活性分析表明,重组VIP能显著降低急性炎症小鼠血清中抵抗素的水平,发挥抗炎作用。重组VIP的制备及其抗炎活性的鉴定为其深入开发奠定了基础。     

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.     

Simultaneous refolding and purification of a recombinant lipase with an intein tag by affinity precipitation with chitosan

A strategy for simultaneous purification and refolding of proteins overexpressed with an intein tag is described. A recombinant lipase overexpressed in Escherichia coli ER2566 with the intein tag and obtained as inclusion bodies was solubilized in buffer containing 8 M urea or cetyltrimethylammonium bromide. The solubilized lipase was precipitated with chitosan and the affinity complex of the polymer with the fusion protein was obtained. The intein tag was cleaved with dithiothreitol and the refolded lipase was obtained in active form. Activity recovery of 80% was observed and the enzyme had a specific activity of 2965 units/mg. The purified lipase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purity and activity recovery were comparable with that of the preparation obtained by using the commercial kit which utilizes chromatography on chitin beads. The purified and refolded lipase was characterized by fluorescence and CD spectroscopy.     

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein-Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein-Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mgGel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 degrees C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.     

Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.     

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.     

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.     

Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing

An intein-mediated approach was developed for expression and affinity purification of a protein that is lethal to Escherichia coli. The protein, I-TevI, is an intron-encoded endonuclease. The approach involved the insertional inactivation of I-TevI with a controllable mini-intein placed in front of a cysteine required for splicing (an I-TevI::intein fusion). The purification was facilitated by a chitin-binding domain inserted into the mini-intein. Affinity purification of the I-TevI::intein fusion precursor on a chitin column was followed by pH-controllable splicing to restore the structure and function of I-TevI. To study the impact of the insertion context on I-TevI inactivation, the chimeric intein was inserted independently in front of seven cysteines of I-TevI. One of the seven intein integrants yielded I-TevI of high activity. This technique is, in principle, generalizable to the expression and purification of other cytotoxic proteins and is amenable to scale-up.     

Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 degrees C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly-Arg-Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies.     

Expression and purification of glycine N-methyltransferases in Escherichia coli

Expression and purification of recombinant mouse, rat, and human glycine N-methyltransferases (GNMTs) in pTYB and pET expression vectors was done in order to prepare the proteins for structure studies of the enzymes from different sources. When human and mouse GNMTs were expressed in pTYB vector as a fusion protein with intein and the chitin binding domain, an unusual cleavage of intein was found. This cleavage takes place at two sites near the N-terminus of intein. This resulted in the appearance of an abnormal GNMT protein after on-column cleavage of the fusion protein, which could not be separated from normal GNMT. For this reason expression of mouse, rat, and human GNMTs was done in the pET-17b expression vector, resulting in the expression of soluble protein at about 20-40mg/L of culture. A new procedure for GNMT isolation after expression in the pET vector was developed. This included only two steps, ammonium sulfate precipitation and ion-exchange chromatography, and resulted in preparations containing 95-97% pure protein. All expressed proteins were tetrameric with molecular weights of 130kDa as determined by size-exclusion chromatography. Activity in Tris buffer at pH 9 of mouse, rat, and human GNMTs was found to be 255, 260, and 540U/mg, respectively. This implies that expressed and purified GNMT proteins are biologically active and suitable for biochemical and structural studies.     

Intein-mediated rapid purification of Cre recombinase

Cre recombinase produced by bacteriophage P1 catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps. In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein. The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column. Using the intein system, we have obtained highly pure nontagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification. The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months.     

Expression of bovine inhibin beta subunit in Escherichia coli.

1. A DNA fragment encoding the beta subunit of bovine inhibin was amplified using the polymerase chain reaction and was cloned in plasmids pUC8 and pUR291. 2. Cultures of Escherichia coli TG2 harbouring pKDK37, a pUR291-derived recombinant plasmid, produced a novel protein with a molecular weight of 130,000 corresponding to a beta-galactosidase-inhibin beta fusion protein. 3. The fusion protein was purified from inclusion bodies by solubilization in 8 M urea followed by an ion-exchange and gel permeation chromatography. 4. Analysis by immunoblotting and competitive radioimmuno assay revealed that the fusion protein was recognized by a monoclonal antibody raised against a chemically synthesized peptide for amino acid residues from +82 to +114 of the beta subunit of the bovine inhibin thereby confirming its identity.