Patent Application: US-201414906887-A

Abstract:
a polypeptide having at least 70 % homology , in particular 80 %, 90 % or 95 % homology to the polypeptide of seq id no : 2 representing the wild - type of the protein insect metalloproteinase inhibitor impiα and having at least one mutation at position 35 , 36 and / or 39 of the amino acid sequence of impiα and the polypeptide having an ic 50 value to thermolysine of less than the ic 50 value of impiα wherein the nonpolar amino acid isoleucine at position 35 of impiα is replaced either by a nonpolar amino acid selected from the group consisting of leucine , methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine , asparagine , glutamine , histidine , lysine and arginine ; and / or the nonpolar amino acid isoleucine at position 36 of impiα is replaced either by a nonpolar amino acid selected from the group consisting of valine , phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine , serine , threonine , asparagine , glutamine , histidine , lysine and arginine ; and / or the polar amino acid position 39 of impiα is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine .

Description:
in one aspect of the invention , biologically active loop polypeptide of the invention polypeptides are provided which exhibit a specificity profile different from the wtimpiα profile for inhibiting members of the m4 protease family . the fact that the loop polypeptide of the invention are biologically active at all is surprising because the loop amino acids were particularly well conserved during evolution . it is even more surprising that altering a specific loop amino acid could increase the binding constant for thermolysin or pseudolysin , or even both . new ways of improving the inhibitory constant by substituting i36 ( isoleucine ) of wtimpiα have been developed , which is surprising since it was assumed that at this position 36 isoleucine would already exhibit a perfect profile for a c - c interaction with amino acids h146 ( histidine ) and t157 ( tyrosine ) of the peptidase , and therefore no attempts were reported to design impi mutants or small molecules with the goal to strengthen this interaction further . the inventors found , however , that a long aliphatic side chain of an amino acid or modified amino acid at this position , preferably with an elevated pka of 12 or higher , improves the inhibitory constant substantially . examples of well suited amino acids comprise k , m , h , and in particular r . surprisingly it was found that further substitutions , for example those listed in the ensuing paragraph , could increase the potency of the inhibitor ( tables 1 , 2 ): loop the polypeptide of the invention molecules exhibiting improved inhibition of thermolysin comprise i35l , i36f , k , r , v , y , q , d , h , m , t , or w , r39k or v . loop the polypeptide of the invention molecules exhibiting improved inhibition of pseudolysin comprise i35l or f , m , w or y , i36r , q , or m , r39k and a . loop polypeptide of the invention molecules exhibiting improved inhibition of both , thermolysin and pseudolysin in parallel comprise i35l , i36r , q , or m , r39k . loop polypeptide of the invention exhibiting equal potency against both , thermolysin and pseudolysin , comprise i35l , or w . this result is surprising because there no reasonable expectation of success if one amino acid is altered close to an area associated with the function of the respective protein . in particular this result is surprising considering the general assumption that a peptidic inhibitor of an enzyme should contain hydrophobic amino acids binding to the enzyme close to its hydrophobic active center ( handbook of proteolytic enzymes 2013 , biela et al . 2013 ). the results show , however , that hydrophobicity of the amino acids in the polypeptide of the invention is not the dominant factor for the inhibition . it is understood that combinations of single site mutations could alter the properties of the polypeptide of the invention even further , and further increase the potency of the inhibitor . the polypeptide of the invention may be obtained by known techniques for directed evolution as described by arnold et al ., 2003a and arnold et al ., 2003b , or may further be obtained by knowledge based engineering of the respective amino acid sequence , including application of rules for conservative or non - conservative amino acid replacement considering size , charge , polarity and other biochemical parameters of the amino acids as know by one of ordinary skill in the art by the teaching and principles disclosed in u . s . pat . nos . 7 , 732 , 587 and 4 , 554 , 101 , or by using suitable computer programs calculating or estimating the binding energy of the mutant impiα and the selected target proteases , by assessing homologies to other proteins or the antigenicity of the protein sequence , and introducing the calculated , modified gene sequence into the bacterial genome by standard molecular biotechnology methods . amino acid deletions within any impiα and its muteins may be made in regions outside the loop region ( aa 35 to 39 of seq id no : 2 ), where they are supposed to not directly interact with the target protease . amino acid deletions may also be made within the loop region , where they are supposed to more likely modify the biological activity . comparison of wtimpiα with similar proteins from other species , such as seq id no : 95 from solenopsis , and 23 other insect related aa sequences depicted in fig9 reveals that the loop region ( aa 33 to 41 in seq id no : 2 , and in particular in region 35 to 39 ) is highly conserved with the exception of aa39 ( r ). it is thus surprising that loop mutein impiα , for example , seq id nos : 10 , 18 , 20 , 22 , 24 , 26 , 28 , 30 , 32 , 34 , 36 , 38 , 40 , 42 , 44 , 46 , 48 , 50 , 52 , 54 , 56 , 58 , 60 , 62 , 64 , 66 , 68 , 70 , 72 , 74 , 76 , 78 , 80 , 82 , 84 also exhibit a strong , undexpected thermolysin inhibiting activity . in another aspect of the present invention it has been found that wtimpiα and mutein impiα , and in particular loop polypeptide of the invention can be recombinantly produced in bacterial cells under conditions identified by the inventors , especially at room temperature and up to 37 ° c ., under conditions which do not require cooling of the fermenter or refolding of the protein produced . the method according to the invention , equipment and reagents used are described in detail in example 2 below . it should be understood , however , that modifications in concentrations of reagents used , and modifications in the genetic configuration of the producer organism ( e . coli ) could be made without affecting the output of correctly folded protein . for example , e . coli could be replaced by other bacteria as production host , additional plasmids or gene stretches with additional function could be added to the producer bacteria . furthermore , medium components could be provided in different order or concentrations , and incubation times could be varied . it should be understood that those modifications which influence the production efficacy of biologically active mutein or wtimpiα negatively by 50 % or less or even positively are considered within the scope of the invention . the invention contemplates further modifications of elements of the expression system inasmuch as the expression of a functional mutein or wtimpiα without the need for refolding is maintained to at least 50 % compared to the system disclosed in example 3 and 4 , and no cooling of the fermenter is required . for example , other expression vectors than pet - 41a (+) may be used , which may not be linked to a fusion tag , or may be linked to a fusion tag other than gst , his - tag ; and other promoters than t7 , and may also include further regulators or other fusion elements . depending on the operons employed , iptg or other inducers may be employed . other restriction sites than pshai may be used in pet - 41a (+) or in other vectors , for example xhoi , ecor i , bamh 1 . other e . coli strains than rosetta - gami 2 plyss may be used , with or without additional t - rna genes or ahp c mutation , and other bacteria than e . coli may also be employed . further modifications may relate to the downstream process steps which are known to the one skilled in the art , including but not limited , to incubation times , reagents and their concentrations and disposables used , for example capture resins and chromatography equipment and separation media for purification . recombinant production of mutein or wtimpiα in bacteria is very cost effective compared to all other production methods for proteins . cloning and the subsequent development of a cell line producing the peptide are faster and less costly with bacteria as , for example , with mammalian or insect cells . during production , the amount of protein produced per volume fermentation broth is much higher compared to eukaryotic cell cultures . consequently there is a strong demand for methods of recombinant protein producing in bacteria , especially in e . coli since they are a proven host frequently used for protein production . it is known that wtimpiα retains its biological activity after deglycosylation ( wedde et al . 1998 ); therefore bacterially produced mutein or wtimpiα lacking glycosylation are biologically active once folded correctly . the skilled artisan would expect , however , that a protein like mutein or wtimpiα exhibiting 5 cystein bridges would not be correctly folded and thus be accumulated almost entirely in inclusion bodies of the e . coli host . while several processes were developed earlier to isolate and dissolve inclusion bodies , and to denature its protein content using chaotropic salts like urea or guanidylchloride , and to withdraw the salts again in an ensuing step , for example by dialysis , to enable refolding of the protein in a kinetically controlled manner . however , the success rate of refolding proteins with multiple cystein bridges , such as as impiα , is usually low , and even today time consuming and resource consuming trial and error methods are required to find adequate refolding conditions . it is generally not yet possible to predict or experimentally determine suitable refolding conditions . it is thus surprising that , without any known exception , a family of cystein rich proteins such as mutein or wtimpiα with a complex tertiary structure can be recombinantly produced in bacteria by the method of invention and without the need for refolding steps , in particular that the production can take place without cooling of the fermenter , the latter being a common measure to reduce the protein production rate of the bacteria and thereby to reduce the ratio of incorrectly folded protein vs . correctly folded protein . in another aspect of the invention , the use of mutein or wtimpiα , and in particular of wtimpiα is provided for inhibition of the anthrax protease nrp599 . current approaches to treat anthrax include vaccination such as biothrax ( emergent biosolutions , rockville md ., u . s . a ) for disease prevention , or antibiotics such as ciprofloxacin or doxycyclin ( national terror alert response center ). while the vaccine is only approved for preventive use , it takes several days until antibiotics exert effects on bacteria to an extent that the symptoms caused by their virulence factors are reduced . compounds neutralizing virulence factors were developed recently and are generally well known in the state of the art and for anthrax most compounds targeting virulence factors developed so far target lf . however , no specific inhibitor of nrp599 was reported so far , although recent evidence shows that nrp599 is one of the major anthrax virulence factors inducing hemorrhagic bleeding , for example ( chung et al ., 2006 ) by targeting the fibrinolytic system ( chung et al , 2011 ), and that it also degrades the van willebrand - factor and thereby interferes with the coagulation cascade ( chung et al ., 2008 ). us 2004 / 018193 teaches the concurrent or offset administration of both , antibiotics and antigenic compounds , in particular to treat anthrax infected humans . the authors provide several examples of protease inhibitors including hypothetical antibodies , but they do not disclose nrp599 as a target . surprisingly it was found that polypeptides of the invention and in particular wt impiα is a potent and specific inhibitor of nrp599 , despite the fact that bacillus anthracis is not known to infect insects . it was found that wtimpiα inhibits npr599 at nanomolar concentrations . to treat anthrax infection or symptoms caused by anthrax toxins , the polypeptide of the invention and in particular wt impiα may be combined with antibiotics , in particular ciprofloxacin or doxycyclin , with antimicrobial peptides , with other inhibitors of bacterial proteases , or with compounds improving the physiological state of the infected patient . the polypeptide of the invention may also be produced effectively in recombinant cell lines and tissue , including mammalian and insect expression systems , plant and bacterial systems ; or synthetically . mutein and wtimpiα each may typically be isolated and purified to be free of other proteins and protein fragments . preferably , mutein and wtimpiα is about 80 % free of other proteins which may be present due to the production technique used in the manufacturing process . more preferably , the polypeptide of the invention or wtimpiα is about 90 % free of other proteins , particularly preferably about 95 % free of other proteins , and most preferably about & gt ; 98 % free of other proteins . it will be appreciated , however , that the desired protein may be combined with other active ingredients , chemical compositions and / or suitable pharmaceutical formulation materials prior to administration as medication , as described in further detail below . the biological activity of mutein or wtimpiα can be assessed in vitro by means of assays , such as measuring binding of the polypeptide of the invention or wtimpiα to a protease immobilized on coated glass carrier used for plasmon resonance spectroscopy . alternatively , the polypeptide of the invention or wtimpiα can be immobilized on the glass carrier and the protease could be added to start the assay . this assay is able to measure on - and off - rates for the respective interaction . the biological activity can also be tested by means of an parallel line assay , for example in a microtiter plate format , and by detecting a coloration effect or by measuring changes in the fluorescence emitted upon excitation with a suitable light source . amino acid sequence additions may further include n - or c - terminal fusions ranging in length from one residue to 150 or more residues , as well as internal intrasequence insertions of single or multiple amino acid residues into mutein or wtimpiα ranging typically from about 1 to 10 amino acids , more typically from 1 to 5 amino acids , and most typical from 1 to 3 amino acids . n - terminal additions include the addition of a methionine or an additional amino acid residue or sequence . another example of n - terminal addition includes the fusion of a signal sequence — provided the signal sequence seq id no : 94 is not bluntly added to wtimpiα of seq id no : 2 . further examples for additions to mutein or wtimpiα comprise fusions with antibodies ( abs ) or antibody fragments . ab fragments may comprise for example scfab , scfv fragments , or single domain antibodies , or antibodies of camelide ( hcig ) or shark ( ignar ) origin , or their respective vhh domains . the fusion may serve to provide a targeting moiety ( the cdr domains ) fused to to mutein or wtimpiα so that the fusion protein could be accumulated and enriched at surface displaying antigens specific for the antibody fragments . another motivation for fusing mutein or wtimpiα to antibodies may be to benefit from the dimerization properties of the constant antibody regions to obtain a bivalent form of mutein or wtimpiα , in a way similar to etanercept ( enbrel ) in which 2 tnf receptor fragments are fused to the constant domains of an antibody . in addition , such bivalent antibody chimera is sufficiently large to escape from rapid renal clearance , for which a size exclusion barrier of roughly 65 kda exists , so that the lifetime of the fusion protein in the patient body is strongly prolonged . additions may also comprise other scaffolds derived , for example , from lipocalin , ankyrin , fibronectin , transferrin , tetranectin , adnectin , albumin , uteroglobin , or protein a . other additions contemplated comprise functional peptides , for example transferrin enabling the fusion protein to cross the blood brain barrier , a property potentially useful for inhibiting bacterial toxins accumulated in the brain or spinal cord , or additions useful for diagnostic applications , such as green fluorescent protein ( gfp ) enabling fluorescence detection , or peptide tags enabling immobilization on technical surfaces specifically the polypeptide of the invention described herein may involve addition or deletion of or substitution with a non - native amino acid at the n - or c - terminus or at any site of the protein that is modified by the addition of an n - linked or o - linked carbohydrate . a cystein , for example , may be added for linking a water soluble polymer such as polyethylene glycol , or other amino acids like lysine , cysteine , histidine , arginine , asparaginic acid , glutamic acid , serine , threonine , or tyrosin could also be used for coupling polymers to the peptide . another example is the insertion of tripeptide sequences nxt or nxs or fragments thereof with x designating any amino acid except p , which may be recognized by a cellular enzyme adding glycosylation elements . suitable , clinically acceptable , water soluble polymers include polyethylenglycol ( peg ) and polysialic acid ( psa ). another mutation or addition may include tags , in particular peptide tags , such as hexa - histidine which can be used to facilitate binding , for example , to a moiety of a purification column . these tags may comprise a peptide sequence serving as a substrate for a protease , so that the tag can be cleaved in purpose once it is not required anymore . further contemplated are mutein or wtimpiα to which chemical compounds or nano - particles are added . this can be achieved for example by employing the enzyme o - 6 - alkylguanin - alkyltransferase ( agt ) or derivatives thereof to mutein or wtimpiα , or to a peptide tag . other examples comprise conjugation to a cysteine being introduced by a mutation ( jagath et al . 2008 ), or to carbohydrate moieties ( fischer - durand et al . 2010 ). chemical compounds may include for example labeling moieties , in particular for in vivo imaging applications , targeting moieties such as receptor ligands to enrich the conjugated impiα family members in specific organs . another compound suited to immobilize conjugated impiα family members on a surface or particle is biotin , for example for diagnostic purposes . nanoparticles such as quantum dots may be added for in vivo detection or imaging applications . dendrimers or others particles increasing the size of the conjugate may be added to prevent the conjugate from cleared rapidly from the blood stream by the kidneys . modifications to the peptide backbone are also contemplated under the invention , such as beta peptides , where the amino group is attached to the beta carbon of the respective amino acid instead of the alpha carbon , rendering them invulnerable to proteases . other modifications of the peptide backbone are possible , such as alkylation of the amid nitrogen and bioisosteric replacement of amide groups . the present invention further provides polynucleotides which contain nucleotide sequences encoding mutein or wtimpiα , provided the polynucleotide is not identical to the complete wild type sequence seq id no : 1 , or seq id no : 1 directly followed by seq id no : 3 . further are considered nucleotide sequences in which oligonucleotide stretches are inserted which do not code for wtimpiα . based upon the present description and using the universal codon table , one of ordinary skill in the art can determine all of the nucleic acid sequences which encode mutein or wtimpiα . presently preferred nucleic acid sequences include those encoding seq id nos : 9 , 17 , 19 , 21 , 23 , 25 , 27 , 29 , 31 , 33 , 35 , 37 , 39 , 41 , 43 , 45 , 47 , 49 , 51 , 53 , 55 , 57 , 59 , 61 , 63 , 65 , 67 , 69 , 71 , 73 , 75 , 77 , 79 , 81 , 83 . chemical synthesis of mutein or wtimpiα is also contemplated , and is the obligatory route for synthesizing modified mutein or wtimpiα containing non - natural amino acids or backbone modifications . while chemical synthesis was traditionally regarded as being too expensive for larger peptides , recent advances in synthesizing technology like microwave assisted synthesis have recently shifted this limit towards larger proteins . recent approaches also solve the issue of correct configuration of currently up to 3 cystein bridges by using several distinct protective groups in parallel . while the invention contemplates the use of e . coli for producing mutein or wtimpiα with high quality and efficiency , other hosts may also be used for recombinantly producing mutein or wtimpiα , including other strains of e . coli , other bacteria like bacillus megaterium , fungal systems like yeast , pichia , or hansenula , eukaryotic cells like protozoa , insect cell lines like schneider t7 or sf9 mammalian animal cell lines like cho , human cell lines like hek293 , or transgenic animals like goats , or plants like tobacco . mutein or wtimpiα or modified versions thereof may be combined with ingredients to form a pharmaceutical composition . the pharmaceutical composition may include water and salts at physiological concentrations , solubilizing or dispersing agents , or anti - oxidant , or particles forming micelles , such as liposomes . liposomes may also contain baimpiα or modified versions thereof internally . this pharmaceutical composition may be filled in a glass or plastic vial , or in a syringe . the pharmaceutical composition may also contain additives supporting drying or freeze - drying of the pharmaceutical composition , for example cyclodextrins or saccharides , in particular disaccharides . mutein or wtimpiα or modified versions thereof may be used as a medicine , in particular as a medicine for parenteral injection , to treat diseases in an animal , in particular in a mammal or bird , or in a human , and which are related to the activity of proteases from the m4 family . such disease could be , for example , a bacterial infection , in particular infection by staphylococcus aureus , in particular multi - resistant staphylococcus aureus , bacillus anthracis , pseudomonas aeruginosa , helicobacter pylori , vibrio cholerae , or legionella pneumophilia . such a disease could further be systemic inflammatory response syndrome ( sirs ), or sepsis . mutein or wtimpiα or modified versions thereof may be administered parenterally , orally , or topically using suitable pharmaceutical compositions , or attached to a patch or wound debridement from where the medication elutes into a wound of the patient . mutein or wtimpiα or modified versions thereof may be administered in combination with other treatments , in parallel or staggered , in particular with other inhibitors of virulence factors , antibiotics , or antimicrobial peptides . further contemplated are rna molecules whose sequences comprise rna sequences of mutein or wtimpiα , in particular sequences like seq id nos : 1 , 5 , 9 , 11 , 17 , 19 , 21 , 23 , 25 , 27 , 29 , 31 , 33 , 35 , 37 , 39 , 41 , 43 , 45 , 47 , 49 , 51 , 53 , 55 , 57 , 59 , 61 , 63 , 65 , 67 , 69 , 71 , 73 , 75 , 77 , 79 , 81 , 83 , 85 , 87 , 89 , 91 , and which can be transcribed into mutein or wtimpiα protein in the target organism . such rna molecules may be administered in combination with transfection agent like liposomes or pei . the invention also contemplates the application of mutein or wtimpiα in devices for diagnostic purposes . in several cases it is beneficial to determine the protease activity in biological fluids , and in particular the protease activity profile if several of them are present . for fluids contaminated with bacteria determining the protease activity would provide information not only about the presence of bacteria , but also of their activity and relative concentrations . in wounds , and especially in slowly healing or chronic wounds , determining the protease activity profile also is of interest since the physiological stages of wound healing are characterized also by a specific evolution of protease activity during healing . it is even more important to distinguish between the activity of endogenous host proteases and the activity of bacterial proteases . pcr or real time pcr assays do not provide sufficient information because these tests quantify the concentration of particular bacteria present , but they are not suited to test for the activity of the bacteria . currently laboratory tests for protease activity exist where the cleaved protease substrate becomes fluorigenic . many of these tests determine overall protease presence ; some of them are able to profile the protease concentrations ( chen et al . 2013 ). currently the only point - of - care test for protease activity , woundchek ® manufactured by systagenix , ( gatwick , groβbritannien ) measures overall protease activity , but cannot quantify the concentration of each protease species separately ( strohal et al . 2012 ). a diagnostic method according to the invention solves this issue . mutein or wtimpiα is used therein to bind m4 proteases specifically . different concentrations of proteases can be determined when more than one type of mutein or wtimpiα is used . the difference in binding strength between the polypeptide of the invention or wtimpiα is then used to calculate differences in concentrations of mutein or wtimpiα . a preferred embodiment of the diagnostic method uses a device similar to lateral flow tests known in the state of the art ; in which a membrane strip ( suagfähig ) is used to take up the fluid sample and to transport the sample across the membrane by capillary forces . in a preferred embodiment , the membrane consists of nitrocellulose or nitrocellulose endorsed by nylon fibres or tissue . at a particular location on that strip , mutein or wtimpiα are immobilized so that at least part of the sample fluid passes across this location during flow . proteases present in the sample are captured by the immobilized mutein or wtimpiα . in a second step one further solution containing detection capture molecules targeted against different binding epitopes of the proteases are added . these detection capture molecules are prepared to deliver a detection signal , which can be , for example , chromophoric , fluorescent , or electrochemical by means of a direct or enzymatic label . it is possible to increase signal strength by secondary capture molecules targeting the detection capture molecule . in a preferred embodiment , at least one kind of capture molecule is an antibody . a particular advantage of the diagnostic method according to the invention is that several proteases can be distinguished by means of specific binding to specific locations where different mutein or wtimpiα are immobilized , while only a single sort of antibody is required . furthermore , a control area is present at a different location on the membrane strip where detection capture molecules or secondary capture molecules bind to even if no proteases are present in the sample . in a preferred embodiment , the capture functionality in the control area is provided by immobilized proteases or immobilized detection capture molecules . in a different embodiment , capture molecules other than mutein or wtimpiα , in particular antibodies are immobilized on the membrane strip and the proteases are added as detection capture molecule , in particular in combination with secondary capture molecules . this embodiment also requires a contral area containing immobilized proteases or mutein or wtimpiα . the assessment of the protease content is made by visual inspection or a sensor device , including cmos based imaging sensor or scanners . in case that sensors are used , a software can be employed to calculate the true concentration ratios of the proteases from potentially overlapping binding profiles of the polypeptide of the invention or wtimpiα if the individual binding constants or other binding parameters are known . in particular such software may use a linear equation system to calculate the individual concentrations . in another preferred embodiment , the binding kinetics are sampled , in particular by using an imaging sensor , to determine concentrations more accurately . the sample may be added , for example , by a pipetting device , a paper strip , a cotton swab or a needle . the device may dispose of a seal preventing uncontrolled flow across the membrane strip . to assist in understanding the present invention , the following examples are included which describe the results of a series of experiments . the following examples relating to this invention should not , of course , be construed in specifically limiting the invention and such variations of the invention , now known or later developed , which would be within the purview of one skilled in the art are considered to fall within the scope of the present invention as hereinafter claimed . last instar larvae were used for immunization using the following solutions : ( 4 . 05 * 10 4 cfu / ml ) escherichia coli strain bl21 ( de3 ) ( invitrogen , carlsbad , calif .) and ( 0 . 7 * 10 4 cfu / ml ) micrococcus luteus ( dmsz reference number 495 ). ten microliters of sample volume from each solution was injected dorsolaterally into the hemocoel using 1 - ml disposable syringes and 0 . 4 - mm needles mounted on a microapplicator . larvae were homogenized at 8 h postinjection for total rna isolation using the tri - zol reagent ( invitrogen , germany ) according to manufacturer &# 39 ; s instructions . cdna was synthesized using the onestep rt - pcr system ( qiagen , germany ) according to the manufacturer &# 39 ; s instructions . the t7 - based expression system , pet - 41a (+), which allows protein expression upon induction of the t7 - polymerase in escherichia coli cells by iptg , was chosen to obtain strong and reproducible expression of the recombinant protein . the sequence of the mature impiα was amplified using the forward primer ( 5 ′- ga - tagtcctaatttgtaacggtggacac - 3 ′) and the reverse primer ( 5 ctac - gaacgtattttaggacagtcttttatcg - 3 ′). the fragment was cloned into the pshai site of vector pet - 41a (+), which was then designated p41impiα and transformed into e . coli rosetta - gami 2 plyss ( novagen , germany ). the resulting clones were verified via sequencing ( eurofins mwg operon , germany ) i . preparatory culture : lb - medium containing 1 % ( w / v ) glucose , cm 34 , kan 50 and tc 12 , 5 was inoculated with cell material from a colony and cultivation started on an orbital shaker at 32 ° c . up to a od 600 of 1 . the preparatory culture was subsequently stored at 4 ° c . in a refrigerator . ii . main culture : lb - medium containing 1 % ( w / v ) glucose , cm 34 , kan 50 , was inoculated with 3 %( v / v ) of the preparatory culture and cultivation started on an orbital shaker at 32 ° c . up to a od 600 of 1 . upon reaching an od 600 of 1 the expression of the recombinant protein was induced with 1 mm iptg . the main culture was subsequently incubated for further 3 hours at 32 ° c . on an orbital shaker . iii . cells were harvested from liquid culture by centrifugation at 10 , 000 g for 10 min using a centrifuge tube of known weight . the pellet was decanted and allowed to drain , removing a maximum amount of liquid before the wet weight of the pellet was determined . iv . the cell pellet was resuspended in bugbuster ® reagent ( novagen , germany ) at room temperature by pipeting and gentle vortexing . 5 ml reagent per g wet cell paste was used . 1 μl ( 25 units ) benzonase ® nuclease ( novagen , germany ) per 1 ml of bugbuster ® reagent was added . v . the suspension obtained in step ( iv ) of this protocol was incubated at room temperature on a shaking platform at a slow setting for 20 min . care was taken that the extract was not viscous at the end of the incubation . vi . insoluble cell debris was removed by centrifugation at 16 , 000 × g for 20 min at 4 ° c . vii . the supernatant was transferred to a fresh tube . the soluble extract was applied directly to gst • bind ™ resin ( novagen , germany ). viii . affinity chromatography was performed according to manufacturer &# 39 ; s instructions ( ge healthcare life sciences . 3 . main culture : lb - medium containing 1 % ( w / v ) glucose , cm 34 , kan 50 was inoculated with 3 % ( v / v ) of the preparatory culture and cultivation started on an orbital shaker at 32 ° c . until an od 600 of 1 was reached . at 1 od the expression of the recombinant protein was induced with 1 mm iptg . the main culture was subsequently incubated for further 3 hours at 32 ° c . on an orbital shaker . protein motifs were identified using smart ( european molecular biology laboratory , heidelberg , germany ) and the conserved domain database from ncbi ( national center for biotechnology information , bethesda , md ., usa ). the signal peptide was predicted using signalp ( center for biological sequence analysis , technical university of denmark ), and the theoretical isolelectric point and molecular weight were predicted using compute pi / mw ( expasy , swiss institute of bioinformatics ). cysteine disulfide bonding state and connectivity prediction was done by using disulfind ( dipartimento di ingegneria dell &# 39 ; informazione , universita di firenze , firenze , italy ). sequence similarity was analysed by blast from ncbi ( national center for biotechnology information , bethesda , md ., usa ). multi - sequence alignment was generated using vector nti 9 . 0 ( invitrogen , germany ). homology modeling was performed by the cphmodels 3 . 0 server ( center for biological sequence analysis , technical university of denmark ) using the swiss - model server ( expasy , swiss institute of bioinformatics ) for structure assessment . all models were energy minimized by using gromos force field ( gromacs , stockholm , sweden ). the subsequent table lists the sequences printed in the ensuing sequence protocol . the leading number denotes the seq id no for the nucleotide sequence , the subsequent even number would denote the seq id no of the respective peptide sequence . adekoya o , sylte i . the thermolysin family ( m4 ) of enzymes : therapeutic and bio - technological potential . chem biol drug des 2009 ; 73 : 7 - 16 . armstrong p b . proteases and protease inhibitors : a balance of activities in host - pathogen interaction . immunobiol 2006 ; 211 : 263 - 81 . arnold f . h ., georgiou g . 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