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1.
The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications. 相似文献
2.
Alejandro Vian Alfonso V. Carrascosa José L. García Estrella Cortés 《Applied and environmental microbiology》1998,64(6):2187-2191
The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a Km value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.β-d-Galactosidase (EC 3.2.1.23) catalyzes the hydrolysis of β-1,4-d-galactosidic linkages. This enzyme is distributed in numerous microorganisms, plants, and animal tissues. The application of β-galactosidase to the hydrolysis of lactose in dairy products, such as milk and cheese whey, has received much attention (7, 21), and in this regard, thermostable β-galactosidases have attracted increasing interest because of their potential usefulness in the industrial processing of lactose-containing products (21). Thermostable enzymes have a number of generally recognized advantages in industrial applications, such as associated chemical resistance and reduced chances of microbial growth at high temperatures (15, 19). Nevertheless, relatively few studies have been conducted on β-galactosidases from thermotolerant or thermophilic bacteria, and as far as we know, only four genes encoding these enzymes have been cloned (5, 10, 11, 13, 16, 18).An important property that has received little attention in the literature is the level of purity of commercial preparations of β-galactosidases, especially with regard to the presence of other enzymes, such as proteases. These contaminants could have a severe impact on the stability of the enzyme, leading to undesirable changes in dairy products during storage (21). To prevent these, a new method was developed to purify the β-galactosidase (LacZ) of Escherichia coli by fusing to its N terminus the choline-binding domain (ChBD) of the pneumococcal autolytic amidase LytA (23). This system allowed the purification of E. coli β-galactosidase in a single step by affinity chromatography on DEAE-cellulose (23). Thus, it appeared interesting to test whether this procedure could also be used in the purification of a thermostable enzyme in order to circumvent contamination problems.This paper reports the molecular characterization of the bgaA gene, encoding the β-galactosidase (BgaA) of Thermus sp. strain T2, and describes the construction of a ChBD-BgaA chimera which retains the biochemical properties of the native enzyme and can be purified in a single chromatographic step. 相似文献
3.
Daniel Bello-Gil Beatriz Maestro Jennifer Fonseca Juan M. Feliu Víctor Climent Jesús M. Sanz 《PloS one》2014,9(1)
We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. 相似文献
4.
Soheila Matroodi Mostafa Motallebi Mohammadreza Zamani Mehdi Moradyar 《World journal of microbiology & biotechnology》2013,29(8):1517-1523
Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi. 相似文献
5.
6.
Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports
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Benevides C. C. Pessela Alejandro Vian Csar Mateo Roberto Fernndez-Lafuente Jos L. García Jos M. Guisn Alfonso V. Carrascosa 《Applied microbiology》2003,69(4):1967-1972
A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports. 相似文献
7.
To avoid the unwanted and random covalent linkage between the cross-linker and enzyme's active site in covalent immobilization, a genetically encoded “aldehyde tag” was introduced into recombinant lipase and applied for the one-step purification and covalent immobilization of this enzyme. The effects of the immobilization time, temperature and the amount of enzyme were investigated, and the thermo-stability of immobilized lipase was also examined. The specific activity and the kcat/Km of the immobilized lipase using aldehyde tag (IL-AT) were 2.50 and 3.02 fold higher, respectively, than those of the traditionally immobilized lipase using glutaraldehyde (IL-GA). The newly immobilized lipase also presented better thermo-stability than the traditionally immobilized one. The results show that the recombinant enzyme could be conveniently immobilized without glutaraldehyde and that the enzyme's active site was well protected. This is a new immobilization method able to avoid glutaraldehyde or 2,4,6-trichloro-1,3,5-triazine as an activating agent. The greener method without hazardous chemicals for the one-step purification and immobilization of an enzyme using a genetically encoded “aldehyde tag” can be exploited for numerous other enzyme purification and immobilization applications. 相似文献
8.
Ömer Şimşek Seba Sabanoğlu Ahmet Hilmi Çon Nihat Karasu Mustafa Akçelik Per E. J. Saris 《Applied microbiology and biotechnology》2013,97(10):4577-4587
In this study, nisin producer Lactococcus lactis strains displaying cell surface chitin-binding domain (ChBD) and capable of immobilizing to chitin flakes were constructed. To obtain ChBD-based cell immobilization, Usp45 signal sequence with ChBD of chitinase A1 enzyme from Bacillus circulans was fused with different lengths of PrtP (153, 344, and 800 aa) or AcmA (242 aa) anchors derived from L. lactis. According to the whole cell ELISA analysis, ChBD was successfully expressed on the surface of L. lactis cells. Scanning electron microscope observations supported the conclusion of the binding analysis that L. lactis cells expressing the ChBD with long PrtP anchor (800 aa) did bind to chitin surfaces more efficiently than cells with the other ChBD anchors. The attained binding affinity of nisin producers for chitin flakes retained them in the fermentation during medium changes and enabled storage for sequential productions. Initial nisin production was stably maintained with many cycles. These results demonstrate that an efficient immobilization of L. lactis cells to chitin is possible for industrial scale repeated cycle or continuous nisin fermentation. 相似文献
9.
Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme 总被引:5,自引:1,他引:4
Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminldases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was cholineindependent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units. 相似文献
10.
Chitin-binding domain (ChBD) of chitinase A1 from Bacillus circulans WL-12 comprises 45 amino acids and exhibits remarkably high specificity to chitin (Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M., Watanabe, T., 2000. Expression and characterization of the chitin-binding domain of chintinase A1 from B. circulans WL-12. J. Bacteriol. 182, 3045-3054.). To investigate the feasibility of exploiting ChBD as affinity tags to confine enzymes of interest on chitin, ChBD fused to the C-terminus of the gene encoding D-hydantoinase was constructed. Subsequent expression of the hybrid protein in Escherichia coli gave a soluble fraction accounting for 8% of total cell protein content. Direct adsorption of the ChBD-fused D-hydantoinase on chitin beads was carried out, and SDS-PAGE analysis showed that the linkage between the fusion protein and the affinity matrix was highly specific, substantially stable, and reversible. As compared to its free counterpart, the immobilized D-hydantoinase exhibited higher tolerance to heat and gained a half life of 270 h at 45 degrees C. In addition, the shelf life (defined as 50% of initial activity remained) of the immobilized enzyme stored at 4 degrees C was found to reach 65 days. Furthermore, D-hydantoinase immobilized on chitin could be reused for 15 times to achieve the conversion yield exceeding 90%. Overall, it illustrates the great usefulness of ChBD for enzyme immobilization. 相似文献
11.
Aimee Shen Patrick J. Lupardus Montse Morell Elizabeth L. Ponder A. Masoud Sadaghiani K. Christopher Garcia Matthew Bogyo 《PloS one》2009,4(12)
We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms. 相似文献
12.
Qiaojuan Yan Aimin Wu Zhengqiang Jiang Luo Tang Shi Bo 《World journal of microbiology & biotechnology》2009,25(6):1057-1063
Cells of the thermophilic Bacillus subtilis WY34 were immobilized on various formaldehyde-activated polymer membranes and the immobilized cells were used for the production
of thermostable mannanase in flasks. The results showed that polyethersulfone membranes (PES) and nylon-6 membranes were the
most suitable supports for cell immobilization to produce the mannanase. Moreover, PES and nylon-6 membranes immobilized cells
provided 1.78- and 1.74-fold higher mannanase activity compared to the control after 4 days of cultivation, respectively.
The immobilized cells on PES and nylon-6 membranes had good stability and retained 131.5 and 114.3% of ability of enzyme production
even after six cycles of repeated batch fermentation, respectively. Active cell growth was observed by scanning electron microscopy
(SEM) after 16 days (four cycles) repeated batch cultivation. Therefore, the membrane-immobilized cells of B. subtilis WY34 can be proposed as an effective biocatalyst for repeated usage for production of the thermostable mannanase. 相似文献
13.
This work describes the genetic engineering and characterization of a histidine-tagged fragment of protein A. The histidine tag results in the site-selective immobilization of the protein A receptor and the preservation of its high ligand affinity when immobilized on solid supports. The fragment was expressed at high yield in E. coli and purified to homogeneity. When selectively immobilized to histidine binding matrices, the protein A fragment exhibits high affinity for soluble IgG. We further demonstrate from adsorption isotherms that the receptor exhibits a homogeneous, high affinity population at densities where steric crowding between large ligands does not affect the apparent receptor affinity. This engineered receptor is appropriate for a range of applications including sensor design or those using immobilized Fc-tagged proteins. 相似文献
14.
Michel Schmidt Nina Tuominen Tove Johansson Stefan A. Weiss Kari Keinänen Christian Oker-Blom 《Protein expression and purification》1998,12(3):323-330
The capsid protein of rubella virus was produced in baculovirus-infectedSpodoptera frugiperdainsect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal–ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen–antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies. 相似文献
15.
de Los Angeles Calixto-Romo M Santiago-Hernández JA Vallejo-Becerra V Amaya-Delgado L del Carmen Montes-Horcasitas M Hidalgo-Lara ME 《Journal of industrial microbiology & biotechnology》2008,35(11):1455-1463
This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD
Cex
from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes
were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively,
were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized
on Avicel. K
m and V
max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent
to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free
form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin,
which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained,
subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating
to V
max increased up to 5.7-fold, following immobilization, whereas K
m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity
was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose. 相似文献
16.
Lipases have found a number of commercial applications. However, thermostable lipase immobilized on nanoparticle is not extensively characterized. In this study, a recombinant thermostable lipase (designated as TtL) from Thermus thermophilus WL was expressed in Escherichia coli and immobilized onto 3-APTES-modified Fe3O4@SiO2 supermagnetic nanoparticles. Based on analyses with tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer observation, the diameter of immobilized lipase nanoparticle was 18.4 (±2.4)?nm, and its saturation magnetization value was 52.3 emu/g. The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field. The biochemical characterizations revealed that, comparing with the free one, the immobilized lipase exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The K m value for the immobilized TtL (2.56 mg/mL) was found to be lower than that of the free one (3.74 mg/mL), showing that the immobilization improved the affinity of lipase for its substrate. In addition, the immobilized TtL exhibited good reusability. It retained more than 79.5 % of its initial activity after reusing for 10 cycles. Therefore, our study presented that the possibility of the efficient reuse of the thermostable lipase immobilized on supermagnetic nanoparticles made it attractive from the viewpoint of practical application. 相似文献
17.
Pessela BC Vian A Mateo C Fernández-Lafuente R García JL Guisán JM Carrascosa AV 《Applied and environmental microbiology》2003,69(4):1967-1972
A novel thermostable chimeric beta-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the beta-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the beta-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports. 相似文献
18.
Preparation and characterization of a thermostable enzyme (Mn-SOD) immobilized on supermagnetic nanoparticles 总被引:1,自引:0,他引:1
Superoxide dismutase (SOD) has been widely applied in medical treatments, cosmetic, food, agriculture, and chemical industries. In industry, the immobilization of enzymes can offer better stability, feasible continuous operations, easy separation and reusing, and significant decrease of the operation costs. However, little attention has focused on the immobilization of the SOD, as well as the immobilization of thermostable enzymes. In this study, the recombinant thermostable manganese superoxide dismutase (Mn-SOD) of Thermus thermophilus wl was purified and covalently immobilized onto supermagnetic 3-APTES-modified Fe(3)O(4)@SiO(2) nanoparticles using glutaraldehyde method to prepare the Mn-SOD bound magnetic nanoparticles. The Mn-SOD nanoparticles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer analysis. The results indicated that the diameter of Mn-SOD nanoparticles was 40 (± 5) nm, and its saturation magnetization value was 27.9 emu/g without remanence or coercivity. By comparison with the free Mn-SOD, it was found that the immobilized Mn-SOD on nanoparticles exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The results showed that the immobilized Mn-SOD on nanoparticles could be reused ten times without significant decrease of enzymatic activity. Therefore, our study presented a novel strategy for the immobilization of thermostable Mn-SOD and for the application of thermostable enzymes. 相似文献
19.
Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails 总被引:1,自引:0,他引:1
Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. (c) 1996 John Wiley & Sons, Inc. 相似文献
20.
《Gene》1997,192(2):271-281
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. 相似文献