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1.
The data on the precursors of bacterial proteases were generalized. The structure and special features of processing of the precursors of bacillary subtilisins, the alpha-lytic protease from Lysobacter enzymogenes and the related chymotrypsin-like proteases from Streptomyces griseus, and the metalloproteases from bacilli and Pseudomonas aeruginosa were discussed. The approaches to producing the precursors and the protease propeptides and to in vitro characterizing them were particularly analyzed. The following physiological functions of the propeptides within the protease precursors were considered probable: (a) inhibition of the proteases to protect the host cells from the proteolytic damage; (b) participation in the folding of the mature enzyme; and (c) providing for the protease interaction with the bacterial cell surveillance mechanisms, including protease translocation through the cell wall. 相似文献
2.
Lise Boon Estefania Ugarte-Berzal Jennifer Vandooren 《Critical reviews in biochemistry and molecular biology》2020,55(2):111-165
AbstractProteases are a diverse group of hydrolytic enzymes, ranging from single-domain catalytic molecules to sophisticated multi-functional macromolecules. Human proteases are divided into five mechanistic classes: aspartate, cysteine, metallo, serine and threonine proteases, based on the catalytic mechanism of hydrolysis. As a protective mechanism against uncontrolled proteolysis, proteases are often produced and secreted as inactive precursors, called zymogens, containing inhibitory N-terminal propeptides. Protease propeptide structures vary considerably in length, ranging from dipeptides and propeptides of about 10 amino acids to complex multifunctional prodomains with hundreds of residues. Interestingly, sequence analysis of the different protease domains has demonstrated that propeptide sequences present higher heterogeneity compared with their catalytic domains. Therefore, we suggest that protease inhibition targeting propeptides might be more specific and have less off-target effects than classical inhibitors. The roles of propeptides, besides keeping protease latency, include correct folding of proteases, compartmentalization, liganding, and functional modulation. Changes in the propeptide sequence, thus, have a tremendous impact on the cognate enzymes. Small modifications of the propeptide sequences modulate the activity of the enzymes, which may be useful as a therapeutic strategy. This review provides an overview of known human proteases, with a focus on the role of their propeptides. We review propeptide functions, activation mechanisms, and possible therapeutic applications. 相似文献
3.
In vivo inhibition of glutamine synthetase (GS) by l-methionine sulfoximine induces sporulation in a protease deficient mutant of Bacillus polymyxa. This induction of sporulation is accompanied by derepression of EDTA insensitive proteases(s) which seems to be specific for differentiation. Some amino acid analogues derepress proteolytic activity without inducing sporulation, but these proteases are sensitive to metal chelators like those in the vegetative cells. When the proteolytic activity is restored, the mutant cells, which are smaller than the parental strain, regain their normal size.Abbreviations GS
glutamine synthetase
- GYS
glucose-yeast extract-salts
- MSO
l-methionine sulfoximine
- Pr
protease deficient mutant
- DON
6-diazo-5-oxo-l-norleucine
- EDTA
ethylene-diaminetetraacetic acid
- EGTA
ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid
- Tris
tris-(hydroxymethyl)-aminomethane 相似文献
4.
S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10?10M, Rh. glutinis acid protease; 1.8×10?7M, Cladosporium acid protease; 3.0×10?6M.These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form. 相似文献
5.
Protease production byBacteroides fragilis ATCC 25285 was determined in batch and continuous cultures. During exponential growth in batch culture, the majority of proteolysis was cell associated. However, as the bacteria reached stationary phase, most of the intracellular proteases were released into the culture medium. Measurements of alkaline phosphatase and -galactosidase, which are respectively periplasmic and cytoplasmic marker enzymes inB. fragilis, showed that secretion of proteases in the stationary phase was a discrete event and was not associated with a general release of cytoplasmic contents. When the bacterium was grown in continuous culture, cell-associated protease activity increased concomitantly with dilution rate (D=0.03–0.23/h). The ratio of intracellular to whole cell protease activity also increased with growth rate (11 at D=0.03/h; 11.7 at D=0.23/h). Extracellular protease activity was detected only in trace amounts in continuous cultures at the lowest dilution rate. Determinations of the distribution of extracellular protease activity in batch culture after 48 h incubation showed that the majority of proteolysis (ca. 90%) was soluble. Nevertheless, a proportion was associated with particulate fractions, which had high specific activities. 相似文献
6.
Yoshifumi Sakata Takaaki Akaike Moritaka Suga Sumiko Ijiri Masayuki Ando Hiroshi Maeda 《Microbiology and immunology》1996,40(6):415-423
To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo. 相似文献
7.
8.
Lee SH Fujita N Tsuruo T 《Apoptosis : an international journal on programmed cell death》1997,2(1):77-83
Reduced thiols (e.g., cysteine) are important in the maintenance of lymphocyte cell viability and growth. L1210 monocytic leukaemia cells were known to have a limited ability to uptake cystine, and they require cysteine for cell growth. L1210 cells underwent apoptosis when cultured without thiol-bearing and dithiol-cleaving compounds, adding thiols suppressed the apoptosis and promoted cell growth. A specific inhibitor of interleukin-1 -converting enzyme (ICE)-like and CPP32-like proteases could suppress L1210 cell apoptosis induced by thiol deprivation. The cell lysates of apoptotic L1210 cells exhibited protease activity that could cleave DEVD-AMC, but not YVAD-AMC, and so CPP32-like proteases, but not ICE-like proteases, were activated and participated in apoptosis. The addition of thiols could suppress CPP32-like protease activation. Although the cell death-suppressor bcl-2-family proteins (bcl-2 and bcl-XL) were recently found to suppress the activation of CPP32-like proteases, the expression levels of death-suppressor bcl-2-family proteins did not change when thiols were added. These results suggest that reduced thiols maintain L1210 cell survival by inhibiting the activation of CPP32-like proteases without changing the anti-apoptotic bcl-2-family protein expression. 相似文献
9.
Sebnem Ozturkoglu Budak Miaomiao Zhou Carlo Brouwer Ad Wiebenga Isabelle Benoit Marcos Di Falco Adrian Tsang Ronald P de Vries 《BMC genomics》2014,15(1)
Background
Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide range of biochemical properties. Several Aspergilli have the ability to produce a variety of proteases, but no comprehensive comparative study has been carried out on protease productivity in this genus so far.Results
We have performed a combined analysis of comparative genomics, proteomics and enzymology tests on seven Aspergillus species grown on wheat bran and sugar beet pulp. Putative proteases were identified by homology search and Pfam domains. These genes were then clusters based on orthology and extracellular proteases were identified by protein subcellular localization prediction. Proteomics was used to identify the secreted enzymes in the cultures, while protease essays with and without inhibitors were performed to determine the overall protease activity per protease class. All this data was then integrated to compare the protease productivities in Aspergilli.Conclusions
Genomes of Aspergillus species contain a similar proportion of protease encoding genes. According to comparative genomics, proteomics and enzymatic experiments serine proteases make up the largest group in the protease spectrum across the species. In general wheat bran gives higher induction of proteases than sugar beet pulp. Interesting differences of protease activity, extracellular enzyme spectrum composition, protein occurrence and abundance were identified for species. By combining in silico and wet-lab experiments, we present the intriguing variety of protease productivity in Aspergilli.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-523) contains supplementary material, which is available to authorized users. 相似文献10.
Regina Fluhrer Harald Steiner Christian Haass 《The Journal of biological chemistry》2009,284(21):13975-13979
Intramembrane-cleaving proteases are required for reverse signaling and
membrane protein degradation. A major class of these proteases is represented
by the GXGD-type aspartyl proteases. GXGD describes a novel
signature sequence that distinguishes these proteases from conventional
aspartyl proteases. Members of the family of the GXGD-type aspartyl
proteases are the Alzheimer disease-related γ-secretase, the signal
peptide peptidases and their homologs, and the bacterial type IV prepilin
peptidases. We will describe the major biochemical and functional properties
of the signal peptide peptidases and their relatives. We then compare these
properties with those of γ-secretase and discuss common mechanisms but
also point out a number of substantial differences.During the last years, a number of intramembrane-cleaving proteases termed
I-CLiPs3 have been
identified (1). I-CLiPs are
generally involved in regulated intramembrane proteolysis
(2). Upon shedding of a large
part of the ectodomain of membrane proteins, the remaining membrane-retained
stub is cleaved by specialized proteases within the hydrophobic lipid
membrane. Generally, this cleavage can have two predominant biological
functions: first, signaling via the liberated ICD within the
substrate-expressing cell (reverse signaling)
(2); and second, degradation of
membrane-retained stubs, which are not required for any further biological
function (3). I-CLiPs of three
protease classes, metalloproteases, serine proteases, and aspartyl proteases,
have been discovered so far (see accompanying minireview by Wolfe
(44)).Intramembrane-cleaving aspartyl proteases are represented by the class of
the GXGD-type proteases
(4). These are unconventional
aspartyl proteases that, like the conventional aspartyl proteases, utilize two
critical aspartyl residues for peptide bond cleavage. However, in contrast to
the conventional proteases, the critical aspartyl residues are located within
two TMDs (Fig. 1A).
Moreover, these aspartyl residues are embedded in active-site motifs that are
completely different from those of conventional aspartyl proteases. The class
of GXGD-type aspartyl proteases is currently represented by three
different protease families, the most prominent of which is the PS family,
providing the catalytically active subunit of γ-secretase
(Fig. 1A)
(4). PS/γ-secretase is
the I-CLiP that liberates amyloid β-peptide, the major component of
senile plaques in Alzheimer disease patients
(5). In addition, the bacterial
type IV prepilin peptidases also belong to the class of the GXGD-type
proteases (6). Besides these
two protease families, two additional subfamilies of related proteases that
also belong to the GXGD-type aspartyl protease family have been
identified. These include SPP as well as the SPP homologs, the
SPP-like (SPPL) proteases
(Fig. 1A)
(7,
8).Open in a separate windowFIGURE 1.A, schematic representation of SPPL2a/b, a member of the SPP/SPPL
family, and PS, the catalytic core of theγ-secretase. Note the opposite
topology of the active sites (indicated by arrows) of the two
proteases and their substrates, APP for PS and TNFα for SPPL2a/b.
B, proteolytic processing of APP and TNFα. Shedding releases
the extracellular part of APP (APPs) and TNFα
(TNFα soluble). In the case of APP, a C-terminal
fragment (APP CTF), and in case of TNFα, an N-terminal fragment
(TNFα NTF) are produced. These membrane-bound
fragments are substrate to intramembrane cleavage by PS or SPPL2a/b,
respectively, releasing small peptides to the extracellular space
(Aβ and TNFα C-domain, respectively) and
to the cytosol (APP intracellular domain (AICD) and
TNFα ICD), respectively). TNFα
FL, full-length TNFα.We will first describe the biochemical, functional, and structural
properties of SPP family members. By comparison of these properties, we will
then identify common mechanisms of intramembrane proteolysis by
GXGD-type proteases but also point out some fundamental
differences. 相似文献
11.
Protease enzymes (proteases), particularly those produced by microorganisms, play very important roles in industry, due to their diverse applications. Considering the richness of microbial diversity in nature, a good chance always exists that proteases more suitable, with better properties for commercial application, may be discovered while screening novel microorganisms from local environments. In this study, 94 yeasts were isolated from different natural sources collected from the Abha region, Kingdom of Saudi Arabia, to determine extracellular protease production and activity. Among them, 23 isolates (24.46%) showed protease activity using a casein hydrolysis test. Of these, five isolates (21.74%) were selected and identified as the best protease producers by exhibiting the largest clearance zones around colonies. A 26S rRNA gene D1/D2 domain sequence alignment, comparison, and phylogenetic analysis of our study yeasts to published D1/D2 domain rRNA gene sequences from GenBank, identifies the isolates as Rhodotorula mucilaginosa KKU-M12c, Cryptococcus albidus KKU-M13c, Pichia membranifaciens KKU-M18c, Hanseniaspora uvarum KKU-M19c, and Candida californica KKU-M20c. The influence of varying pH (4.0–9.0) on the yield and activity of the proteases was investigated using 0.5% (w/v) casein as a substrate, to detect optimum pH values for yeast extracellular protease production. Enzyme activity was measured using qualitative and quantitative assays. Results show all of the study yeasts secreting protease enzyme at all tested pH levels, with the exception of pH 9.0. This indicates that none of the five yeasts are alkaline protease producers. Maximum protease activity (187 U/mL) was observed in strain H. uvarum KKU-M19c at pH 6.0 (only), indicating that strain KKU-M19c only produces neutral protease. The other four yeast isolates, R. mucilaginosa KKU-M12c, C. albidus KKU-M13c, P. membranifaciens KKU-M18c, and C. californica KKU-M20c, produced both acidic (at pH 4.0) and neutral (at pH 6.0 and 7.0) proteases. Strain C. californica KKU-M20c was found to be the best acidic and neutral protease producer (138 U/mL at pH 4.0, and 185 U/mL at pH 7.0). This is the first report of the discovery and isolation of local, powerful yeasts producing acidic and neutral protease enzymes from the Abha region, Kingdom of Saudi Arabia. 相似文献
12.
- Cell-free extracts from vegetative cells and developing myxospores of Myxococcus xanthus were found to contain similar amounts of proteolytic activity, approximately 80% of which was due to one or more neutral metal proteases.
- Sixty per cent of the proteolytic activity was particulate.
- The specific activity of the proteases was high throughout all stages of myxospore formation and displayed small increases in activity at two stages of development: (1) during cell shortening and (2) immediately following the conversion to spheres. The first peak in activity was apparent in assays conducted at pH 8 or 10 whereas the second peak was obvious only at pH 6.
- A mutant which develops into myxospores only after a lag of approximately 7–8 h possessed levels of proteases similar to the wild type and displayed a peak in proteolytic activity after a delay of 7–8 h.
- Low levels of serine protease activity were occasionally detected in both vegetative cells and myxospores; no sulfhydryl proteases were detectable in either cell type.
- Extracellular proteases accumulated in the medium throughout myxospore development but differed from the intracellular proteases in pH optima and sensitivity to inhibitors.
13.
Previn Naicker Ikechukwu Achilonu Sylvia Fanucchi Manuel Fernandes Mahmoud A.A. Ibrahim Heini W. Dirr 《Journal of biomolecular structure & dynamics》2013,31(12):1370-1380
The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7?Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs.An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:36 相似文献
14.
Jurjen Frankena Henk W. van Verseveld Adriaan H. Stouthamer 《Applied microbiology and biotechnology》1985,22(3):169-176
Summary
Bacillus licheniformis S 1684 is able to produce an alkaline serine protease exocellularly. In glucose-limited chemostat cultures the specific rate of protease production was maximal at a -value of 0.22. Above this growth rate protease production was repressed. Dependent on 10–20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products
was 4.1.Maximum molar growth yields were high and indicate a high efficiency of growth. The values of Y
glu
max
and YO
2
max
were 83.8 and 53.3, respectively. When Y
glu
max
was corrected for the amount of glucose used for product formation a value of 100.3 was obtained. These high maximum molar growth yields are most probably caused by a high Y
ATP
max
. Anaerobic batch experiments showed a Y
ATP of 14.6.Sometimes the used strain was instable in cell morphology and protease production. Non-protease producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells most probably are relaxed (rel
-) and non-producing cells stringent (rel
+).Glossary
specific growth rate (h-1)
-
Y
sub
growth yield permol substrate (g biomass/mol)
-
Y
max
maximum molar growth yield, corrected for maintenance requirements (g biomass/mol)
-
Y
max(corr)
Y
max corrected for product formation (g biomass/mol)
-
m
sub
maintenance requirements (mol/g biomass·h)
-
m
sub(corr)
maintenance requirements corrected for product formation (mol/g biomass·h)
-
Y
c
fraction of organic substrate converted in biomass
-
z
fraction of organic substrate converted in exocellular products
-
d
fraction of organic substrate converted in CO2 (g mol/g atom C)
-
Crec%
carbon recovery %
-
average degree of reduction of exocellular products
-
P/O
amount of ATP produced during electron-transport of 2 electrons to oxygen 相似文献
15.
16.
Mark J. Smyth Mark D. Hulett Kevin Y. T. Thia Howard A. Young Thomas J. Sayers Clive R. D. Carter Joseph A. Trapani 《Immunogenetics》1995,42(2):101-111
Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 M
r
serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number L38482. 相似文献
17.
Sharon J. Reid J. Ann Sugrue Jennifer A. Thomson 《Applied microbiology and biotechnology》1986,24(4):311-318
Summary Genes for the -amylase and neutral protease were cloned from an industrial Bacillus isolate, Bsl, onto two separate plasmids and introduced into a B. subtilis strain. Both plasmids were stably maintained in this strain. Analysis of the extracellular proteins showed that the plasmidcarrying strain produced predominantly the Bsl -amylase and neutral protease with few contaminating B. subtilis exoenzymes. The presence of high levels of protease enabled the strain to produce considerably more -amylase when grown on a complex industrial medium rich in protein. 相似文献
18.
Nilegaonkar S.S. Kanekar P.P. Sarnaik S.S. Kelkar A.S. 《World journal of microbiology & biotechnology》2002,18(8):785-789
An extracellular protease was produced by Arthrobacter ramosus isolated from the alkaline lake of Lonar, Buldhana District of Maharashtra, India when grown on a synthetic medium of pH 10 containing casein. The optimum conditions for production were 3.0% initial casein concentration, 2% inoculum of 1 × 108 cells/ml, pH 9.0, temperature 30 °C and shaken culture conditions. The protease was purified by ammonium sulphate precipitation followed by Sephadex G-100 chromatography. Two proteases viz. Arthro I and Arthro II, having molecular weights 21 and 11.4 kDa respectively were isolated. The Arthro II fraction had K
m 395 g/ml and V
max 10.55 g/min for azocasein. The maximum activity of enzyme was at 55 °C and pH 8. It was thermostable (up to 80 °C), alkali stable (pH 12) and stable in commercial detergent. The enzyme may contain a thiol group at the active site. 相似文献
19.
Katsumi Ajisaka Mariko Miyasato Chikako Ito Yasuko Fujita Yoshimitu Yamazaki Syuichi Oka 《Glycoconjugate journal》2001,18(4):301-308
Various O-linked and N-linked sugar chains were linked enzymatically to a fragment peptide (Leu-Ser-Gln(or Asn)-Val-His-Arg) of FGF-5S. First, galactose was linked with -(13)-linkage to GalNAc-linked peptide by a transglycosylation using -galactosidase from Bacillus circulans (recombinant). Then sialic acid was linked with the aid of sialyltransferase from rat liver (recombinant) to give NeuAc-(23)-Gal-(13)-GalNAc-linked hexapeptide. Further, a sialylated 2-chain biantennary sugar chain was linked by a transglycosylation using endo N-acetyl--D-glucosaminidase from Mucor hiemalis (endo M, recombinant). The activity of DNA synthesis in a fibroblast cell line was increased by this glycosylation. The resistance of the obtained glycopeptides towards proteolytic hydrolysis by rat serum and by five proteases was compared with that of original peptide. The resistance was remarkably enhanced by the glycosylation. 相似文献
20.
S. Sonezaki A. Kondo T. Oba Y. Ishii Y. Kato H. Nakayama 《Applied microbiology and biotechnology》1994,42(2-3):313-318
Lon protease, which plays a major role in degradation of abnormal proteins inEscherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form inE. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl--d-methoxynaphthylamide) and protein (-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes. 相似文献