Patent Application: US-16844902-A

Abstract:
the invention provides a vector for expressing a heterologous gene encoding a polypeptide of interest in a gram - negative prokaryote and targeting the expressed polypeptide to the outer membrane and / or periplasmic space thereof , the vector comprising nucleic acid encoding a stromal targeting domain . also provided are host cells containing the vectors , compositions containing the host cells , and the uses of the compositions and host cells .

Description:
expression and targeting of prssu to the om in escherichia coli the precursor of the small subunit of rubisco ( ribulose1 , 5 - bisphosphate carboxyl / oxygenase ) ( prssu ) was expressed in e . coli using the tightly - regulated gp l promoter . e . coli n4830 - 1 cultures , pre - grown in luria broth ( ampicillin 75 mglrnl ) at 30 ° c . to an o . d .= 0 . 6 units , were thermo - induced at 39 ± 1 ° c . for durations specified elsewhere . e . coli subcellular fractionations were performed as described previously ( karim et al ( 1993 ) bio - technology 11 , 612 - 617 ). proteins were analysed by sds electophoresis using either 12 - 18 % gradient of polyacrylamide gels with sample loadings ranging from 50 to 100 μg per lane . for western blots , eletrophoresed proteins were transferred from an unstained gel into nitrocellulose sheets and the rabbit anti - pea mature small subunit of rubisco ( ssu ) reactive components detected by activity staining with horseradish peroxidase conjugated to goat anti - rabbit igg . the membrane - associated prssu , isolated by electroelution , was microsequenced by edman degradation ( alta laboratories , birmingham university ). the periplasmic anti - ssu reactive 14 kda was isolated by electroelution following filtration of a ten - fold concentrated periplasmic fraction through an amicon 30 filter unit . for immunoelectron microscopy , prefixed ulain sections of e . coli were treated with 0 . 5 % bovine serum albunnn , 0 . 2 % gelatin in phosphate - buffered saline to block non - specific binding . after incubation with affinity - purified anti - ssu antibodies ( 1 : 500 ) the sections were extensively washed and labelled with 10 nm protein a - coupled colloidal gold particles , essentially as recommended by biocell conjugates ( uk ). post fixation in osmium tetroxide , the sections were stained with 2 % uranyl acetate and lead citrate ( 18 ) and examined using a jeol jem - 100 cx transmission electron microscope at 100 kv . the 5 ′- proximally - modified prssu cdna , containing an optimised ribosomal binding site and a choice of codons ideal for expression in e . coli was placed under the control of the thezmoinducible xpl promoter in the derivative plasmid pyps ( fig1 a ). the tandemly co - expressed cytochrome b 5 gene , placed downstream of prssu , aided identification and isolation of the clone that expressed prssu through the pink reporter system ( kaderbhai et al . ( 1992 ) dna and cell biology 11 , 567 - 577 ). thermoinduction of e . coli pyps directed the synthesis of two proteins of 20 and 12 kda ( fig2 ). the latter , identified as the co - expressed cytochrome b 5 ( see below ), constitud approximately 9 % of the total cellular protein ( fig2 a , cf lanes t + and t −). whereas the former appeared to represent a sigaificantly smaller amount (˜ 1 %) of the total protein , its detection by coomassic blue staining proved possible only when the total cellular fractions was separated on a ( 15 - 18 %) gradient polyacrylanide gel that provided higher resolving capability ( fig2 c ). the total cellular polypeptide , profile , probed with anti - pea ssu antibodies , signalled cross - reactivity against the 20 kda induced band and , to a lesser extent , with another 14 kda protein ( fig2 b , cf lanes t + and t −). an electroeluted preparation of the 20 kda recombinant protein ( see , materials and methods ) was subjected to 35 rounds of automated edman degradations . this yielded an n - terminal sequence which was identical to that deduced from the nueleotide sequence of the prssu edna , except for the absence of the initiator methionine and the − 15 arginine residue in the transit peptide . the absence of the formylmethionine initiator suggested that the prssu was processed in accordance with the substrate specificity of the cytoplasmic methionine aminopeptidase of e . coli ( hirel et al ( 1989 ) pnas 86 , 8247 - 8251 ). to decipher the sub - cellular location of the prssu and the anti - ssu reactive 14 kda proteins , thermo - induced and non - induced e . coli pyps cells were subfractionated into the periplasmic , cytoplasmic and envelope fractions . the effective separation of the bacterial compartments was confirmed by enrichment of the known marker enzyme activities in the isolated cellular fractions and almost complete recovery of the co - expressed cytochrome b 5 ( table 1 ). the cellular pool of prssu appeared enriched in the envelope membranes ( fig2 a , b , lane e +). the prssu protein proved undetectable in the periplasmic and the cytoplasmic fractions ( fig2 a , b , lanes p +, pd + and c +). the thermoinduced profile of the envelope membranes revealed another dominant , co - expressed 17 kda polypeptide that did not cross - react with the anti - ssu serum . the anti - ssu reactive 14 kda protein localised in the periplasmic fraction ( fig2 b , lanes p + and pd +) was of a size similar to pea stromal ssu ( fig2 a , b , cf lanes p + and s ). moreover , the determined n - terminal sequence of the first five amino acid residues ( mqvwp ) matched with the mature ssu sequence . to test the possibility that the recombinant prssu may have been accumulated in the cytoplasm of the intact bacterium in the form of inclusion bodies which co - isolated with the membranes during subcellular fractionation , a detailed timesourse analysis was conducted by electron microscopy of the thermo - induced e . coli pyps in comparison with the cell - line pλ - lcyt ( gallagher et at . ( 1992 ), applied microbiology and biotechnology 38 , 77 - 83 ) expressing cytochrome b 5 but not prssu . this study showed that both strains had normal ultracytomozphology throughout the induction phase of up to 8 hours ( data not presented ). morphologically , these two recombinant strains were indistinguishable in their cell shape , size and distribution of the nucleoid . the absence of cytoplasmic protein aggregates discounted the likelihood of prssu protein being accumulated in the form of inclusion bodies . unequivocal evidence for the localisation of prssu protein in the envelope zone was obtained by immuno - gold labelling of whole e . coli pyps cell ulstrasections . whilst the izmuno - gold labelling clearly showed that the prssu protein was targeted to the envelope zone , the approach did not indicate whether it was enriched in the im or onl to gain further insight , the cell envelope fraction of thermo - induced e . coli pyps was further resolved into the im and om fractions by discontinuous sucrose gradient centrifugation of the total membrane fraction , obtained by mild lysozyme digestion of edta - treated generated spheroplasts ( osborn et al ( 1972 ) journal of biological chemistry 247 , 3962 - 3972 ). the envelope fraction resolved into two discrete bands with buoyant densities of r = 1 . 23 ± 0 . 02 for the lower white band ( om ) and 1 . 14 ± 0 . 03 for the upper brown band ( im ), values in close agreement with those previously reported . greater than 85 % of the total succinate dehydrogenase activity in the inner membrane and characteristic polypeptide profiles displayed by the two types of membranes ( fig3 a ) substantiated that effective subfractionation of the two membranes had occurred . in the latter , the major of the outer membrane proteins ompa , f and c appeared as the prominent bands but were absent in the im . comparison of the immunoelectrophoretogram in fig3 clearly shows that the envelope - localised prssu was discretely segregated in the om and was undetectable in the im . detectable build - up of prssu in the isolated om occurs at a longer ( 6 h ) induction duration ( fig3 ) than that seen at 4 h in the crude envelope fraction ( fig2 a , b , lane e +), possibly due to loss of some of the precursor protein during the lengthy subfractionation procedures . localisation of prssu protein to the om is concomitantly coupled with induction of two additional proteins of 15 kda and 17 kda . these proteins are not cross - reactive with anti - ssu sera and do not appear in the control thermoinduced cell line px - lcyt . that the prssu protein was tightly integrated into the om was indicated by the inability to extract it from the isolated membranes by washings with either 0 . 1 m na 2 co 3 or 1 m nacl ( data not presented ), treatments known to release loosely - bound and peripheral proteins . to seek whether the prssu protein was laterally exposed to the exterior of the cells , sh thermoinduced , non - permeabilised , e . coli pyps cells were treated with trypsin . whilst , this ‘ shaving ’ approach specifically depleted a 35 kda band , the prssu band remained unaffected , implying that it was most likely not exposed to the exterior to be susceptible to the exogenous protease ( data not presented ). the above experiments demonstrate targeted expression of the pea prssu protein to the om in e . coli . to some extent , the protein is also processed en route to generate a counterpart size related to the mature form in the periplasm . following cytoplasmic synthesis and removal of the n - terminal methionine , frssu was rapidly targeted to the om . translocation to the om could occur either directly from the cytosol via the contact zones or in consecutive steps through the im and periplasm . the former pathway could account for the prssu absence in the im whereas the latter pathway could explain the presences of the processed derivative in the periplasm . in view of the recent findings that the auxiliary periplasmic molecular chaperones are involved in the transit of unfolded im - translocated om proteins to the om ( matsuyama et al . ( 1995 ), the embo journal 14 , 3365 - 3372 ), it is possible that the chromosomally co - expressed 15 kda and 17 kda proteins may be involved in the targeting pathway ( at least in the case of the export of prssu ). expression and targeting of cytochrome b 5 to the om in escherichia coli the ssu portion in the higher plant precursor gene was substituted with the mammalian globular cytochrome b 5 gene in the pyps vector . the dna encoding the 99 amino acid residue globular haemoprotein was placed in a direct reading frame with the transit peptide of ssu ( fig1 b ). the results of this expression study were similar to those observed with the prssu . a significant proportion of processed globular cytochrome b 5 was localised in the periplasm and the chromogenic chimeric transit peptide - cytochrome b 5 was also targeted to the om where it was retained as an integral , correctly folded holoprotein as indicated by its spectral properties ( see fig4 and 5 ). the om - targeted transit peptide - cytochrome b 5 displayed indistinguishable spectral characteristics in comparison with the native cytochrome b 5 , including the soret absorption peak at 423 ran and the visible peaks at 555 nm and 527 nm . thus , the a deleted transit peptide carries targeting information for localisation of a passenger protein to the om of e . coli . most chloroplast transit peptides are particularly rich in hydroxylated amino acids and contain at least several evenly distributed , basic residues ( keegstra ( 1989 ), cell 56 , 247 - 253 ). hence , they are considered to be more soluble in an aqueous environment than the corresponding hydrophobic secretory signal sequences . however , hydropathy analysis of the arginine - deleted transit peptide reveals two hydrophobic regions in the transit peptide , a shorter portion at the amino terminus ( i ) and a longer middle segment ( ii ). albeit displaying a lower hydrophobicity index , the intragenic region ii comprising of 21 residues displays characteristics similar to signal sequences found in the om proteins of e . coli ( fig6 ). these features include an n - terminal region carrying a positively - charged residue , a central hydrophobic core and a c - terminal segment which contains a proline residue located some six residues from a plausible cleavage site according to the − 3 , − 1 rule of von heijne ( heijne ( 1990 ), journlal of membrane biology , 195 - 201 ). similarly , neilson and co - worker &# 39 ; s signalp program ( nielsen et al ( 1997 ) protein engineering 10 , 1 - 6 ) predicts a potential signal sequence in the transit peptide port on spanning from 1 - 21 residues with a potential cleavage site between residues 21 and 22 : qsa - ay . such an n - terminal cleavage or non - cleavable signal sequence segment of the pea transit peptide could act as a membrane insertion loop to initiate the translocation of the passenger polypeptide , possibly by the sec - dependent translocation apparatus . some of the subsequently translocated prssu may have undergone proteolysis to yield a ‘ trimmed ’ form related to the mature ssu in the periplasm . precisely , how the transit peptide subsequently partitions into the om remains to be elucidated and the presence of an additional sorting signal cannot be ruled out . although the particular transit peptide used in the above experiments has a deletion between domains i and ii of an arginine that is highly conserved in most higher plants , the prssu transit peptides of silene pratensis ( swiss - prot entry q42516 ) and amaranthus hypochondriacus ( swiss - prot entry q42516 ) are also devoid of this basic residue . moreover , the arginine - deleted mutant prssu is also importompetent into isolated pea chloroplasts ( data not presented ). targeting of active human cytochrome p4501a1 ( cyp1a1 ) to the periilasmic space of escherichia coli native human cytochrome p4501a1 ( cyp1a1 ) was appended at its amino terminus to the secretory signal of escherichia coli alkaline phosphatase . the chimeric p450 construct was placed under the transcriptional control of the native phoa promoter in a prokaryotic expression vector . induction of the hemoprotein by heterologous expression in e . coli following growth in a phosphate - limited medium resulted in abundant synthesis of recombinant cyp1a1 as detected by reduced co - difference spectra . furthermore , the signal - appended cyp1a1 was translocated across the bacterial inner membrane by the sec - dependent pathway and processed to yield authentic , heme - incorporated p450 within the periplasmic space . in vitro and wholecell metabolic activity studies showed that the periplasmically - located cyp1a1 competently catalysed nadph - dependent benzo [ a ] pyrene 3 - hydroxylation and 7 - ethoxyresorufin o - deethylation . the means to localise cytochromes p450 in the periplasm offers an ability to produce high levels of protein , attributable to the less hostile nature of the compartment and therein the enzymes for post - translational assembly of heme with the translocated protein . cytochromes p450 ( cyp ) are a superfamily of enzymes that are widely distributed in various forms of life including bacteria , plants , fungi , insets and mammals and which perform many important biological oxidations [ 1 ]. cyps contain an iron protoporhyrin ix prosthetic group which catalyses the cleavage of bound molecular oxygen and results in the formation of an oxygenated product [ 2 ]. a plethora of biological reactions are catalysed by cyps including highly selective regio - and stereo specific hydroxylations resulting in commercially relevant products including pharmaceutical and pesticide precursors [ 3 , 4 ]. its unique catalytic chemistry is well known and has been exploited in whole - cell biotransformations , for example , the hydroxylation of corticosteroids by fungi [ 5 ]. however , improvement of biological systems to fully exploit cyps for the synthesis of highly specific chemicals and detoxifying environmental pollutants remains a clear challenge in the areas of biocatalysis and bioremediation . currently , bacterial expression of microsomal human cyps is generally undertaken intracellularly , often localising in the membrane through the cyp n - terminal membrane anchor . expression in escherichia coli often necessitates the modification of the initial part of the open - reading frame to optimise or allow production , therefore producing non - authentic protein . others have reported the use of pelb and ompa leader sequences for cyp production , but successful translocation and processing was not observed [ 6 , 7 ]. in some cases soluble derivatives have also been produced that have allowed the first structural information on these proteins to be obtained after successful production of protein crystals [ 8 ], previously hindered by the hydrophobic n - terminal anchor . for the useful production and study of cyps , higher yields of proteins are desirable , authentic protein production and circumventing permeability problems associated with the cell wall barrier in e . coli . here we utilise the phoa promoter and leader sequence to successfully transport cypial to the periplasmic space in e . coli , where an extremely high yield was obtained . catalytic activity of the protein was observed in an nadphdependent manner reflecting the endogenous electron donor system ( s ) present in this cellular compartment 2 . 1 bacteria and plasmids . the e . coli strains used were dh5αf ′ [ f ′/ enda1 hsdr17 ( r k − m k + ) supe44 thi - 1 reca1 gra96 ( nal r ) rela1 δ ( laczya - argf ) u169 deor φ80dlacδ ( lacz ) m15 ] and tb - 1 [ f , ara ( lac - proab ) rps φ80d laczδm1hsdr17 ( r k + m k + . bacteria were propagated in luria broth composed of 1 %( w / v ) tryptone ( difco ), 0 . 5 %( w / v ) yeast exhact ( difco ), 0 . 5 %( w / v ) sodium chloride and supplemented with 100 μg / ml ampicillin . cyp1a1 production was induced by growth of e . coli in phosphate - limited ( 0 . 1 mm ) mops medium [ 9 ] in the presence of 100 μg / ml ampicillin at 37 ° c . for specified periods ; a saturated lb - culture was used as 2 starter inoculum at 2 %( v / v ). 2 . 2 dna manipulations . plasmid dna was isolated by the wizard midiprep dna purification system from promega the standard procedures for dna restriction , phosphorylation , agarose - gel electrophoresis , preparation of competent cells and transformation of competent e . coli cells were performed as described by maniatis et al ., [ 10 ]. polymerase chain reaction ( pcr ) was performed on an hybaid omnigene thermal cycler , using supertaq ( kramel biotechnology , cramlington , northumberland , uk .). synthetic oligonucleotides were purchased from mwg biotech ( milton keynes , u . k .). plasmid pck1 , which contains the cdna - for cyp1a1 , was used as the template to amplify and manipulate cyp1a1 . the gene was amplified as a pcr fragment of approximately 1500 bp containing the engineered hindiii and psti sites at 5 ′ and 3 ′ ends , respectively , using the following forward and reverse primers : forward primer 5 ′- gctcaagcttcaatggctttcccaatctcc - 3 ′; reverse primer 5 ′- gcgctgcagctaagagcgcagctgcatt - 3 ′. the pcr conditions were as described previously [ 11 ]. the resulting cyp1a1 dna fragment was double digested with hindiii and psti and ligated into the hindiii and psti - cut pliq e . coli expression plasmid to produce plicyp1a1 . the restriction and dna modifying enzymes were purchased from promega ( southampton ) and their conditions for use followed as recommended by the supplier . 2 . 3 subcellular factions . unless otherwise stated , all procedures were carried out at 4 ° c . bacteria induced for heterologous expression ( 500 ml ) were harvested after 20 h by centrifugation at 1500 × g for 10 min . periplasmic fractions were prepared by an ‘ osmotic shock ’ method as follows ; the cells were plasmqlysed by suspension in 2cma 20 %( w / v ) sucrose , 0 . 3m tris - hci ( ph 8 ), 1 mm edta ( ste buffer ) and incubated at 22 ° c . for 10 min . cells were harvested and resuspended in residual ste and osmotically shocked by rapid immersion in 2 ml of ice chilled 0 . 5 mm mgcl 2 . after incubation on ice for 10 min , the perplasmic fraction was recovered by centrifigation at 15000 × g for 10 min . the pellet was retained to provide the material for the preparation of cytoplasmic and membrane fractions . the residual cells , resuspended in 10 ml 50 mm tris - hcl ( ph 8 ), 5 mm na 2 edta containing 10 mg lysozyme were lysed by incubating on ice for 30 min and sonication to reduce the gelatinous chromosomal dna , the lysate made up to 10 mm mgcl 2 , was further incubated for 30 min with 0 . 2 mg / ml dnase i . centrifugation at 100000 × g for 30 min separatd the cytoplasm from the membranes which were prepared as described previously [ 9 ]. 2 . 4 spectrophotometric assays . light - absorption spectra were measured using a hitatchi u3010 scanning spectrophotometer . extracts containing p450 were diluted in 100 mm kpi ( p1h 7 . 4 ) containing 1 mm edta and 20 % ( vlv ) glycerol . p450 concentration was estimated from co reduced difference spectra according to omum and sato , [ 12 ], using an extinction coefficient of 91 mm cmrn . 2 . 5 reconstitution of enzyme activities . in vitro enzyme activities were reconstituted in a reaction mixture ( 1 ml final volume ) consisting of the appropriate e coli periplasmic and cytoplasmic fractions containing 200 pmol of recombinant cyplai . in vivo enzyme activities of 1 ml of e . coli culture harboring the control and cyp1a1 expression plasmids were assessed for enzymatic activity following incubation with substrate . 7 - ethoxyresorufin o - deethylation was estimated flurometrically , using an extraction procedure as described previously [ 13 ]. benzo [ a ] pyrene 3 - hydroxylation was measured fluorometrically according to the method of nebert and gelboin [ 14 ]. 2 . 6 other procedures and assays . protein concentration was estimated using the bicinchoninic acid ( bca , sigma ) assay with bovine serum albumin as standard . sds - polyacrylamide gel electrophoresis employed the discontinuous buffer system of laemmli , [ 15 ]. western blotting was carried out according to the method of guengerich et al ., [ 16 ]. western immunoblots were developed with polyclonal antibody against cyp1a1 ( purchased from gentest corporation , ca , usa ) using alkaline phosphatase for detection . 3 . 1 expression of cyp1a1 in the periplasmic fractions of e . coli . we report here an expression system for the targeting of cytochrome p450 to the periplasmic space of e . coli using the plasmid plicyp1a1 . previous studies for the successful expression of eukaryotic cyps in e . coli have required modification at the n terminus of the p450 or require a leader sequence 5 ′ to the p450 cdna . in the present study , the cyp1a1 . cdna was placed under the tight transcriptional control of the phoa promoter and expression induced by growth of the bacteria in a phosphateulimited medium . lysates of induced bacteria displayed the characteristic reduced co - difference absorption spectrum with a spectral maximum of 447 nm for the expressed native cyp1a1 . the progressive increase in soret absorbance with culture growth indicated that the intensifyug red colour of the induced bacteria was derived from de novo synthesis of cyp . 3 . 2 cyp1a1 targeted to the periplasm of e . coli . subcellular localisation of the recombinant cyp1a1 was investigated and bacteria were subfractionated into periplasmic , cytoplasmic and membrane fractions , with each fraction assayed for cyp content . cyp1a1 was efficiently expressed in the periplasm . at best , 4500 nmol cyp1a1 / l culture was obtained compared with 25 nmol / l culture following n - terminal modification and expression in membranes as described previously [ 11 ]. the effective subcellular targeting to the periplasm was supported by the & gt ; 90 % enrichment of alkaline phosphatase ( periplasm ) and by checking different fractions for malate dehydrogenase ( membranes ) and fumarase ( cytosol ). more than 80 % of the total cellular cyp1a1 content was found localised in the periplasmic fraction of e . coli . further substantiation of the cellular location of cyp forms were sought by subjecting the periplasmic protein factions derived from e . coli tb1 expressing cyp1a1 to sds polyacrylamide gel electrophoresis and western blotting . the identity of the full - length protein was verified by western blot analysis probed with specific anti - cyp1a1 antibodies . 3 . 3 periplasmically targeted cyp1a1 is enzymatically active in vitro and in vivo the catalytic activities of heterologously expressed cyp1a1 were measured in vitro and in whole cell biotransformations . the in vitro activities of cyp1a1 in the isolated periplasmic fractions were determined as a function of varying amounts of the extract . the enzymatic rates of the protein were proportional to the amount of extract used in the , assays . no activity was recorded in control samples derived from e . coli tb1 harbouring the empty plasmid , pliq . the data obtained demonstrated that the activity of native cyp1a1 was comparable to previous reports for these activities of e . coli membranes heterologously expressing cyp1a1 , but which contained modification of the nh2 - terminal domain to allow expression [ 11 ]. the ability of the isolated periplasmic fractions to sustain the biotransformation capabilities of targeted cyp1a1 in the present study strongly suggests that this compartment contains suitable p450 electron donor protes ; previously recombinant mammalian cyps produced within e . coli could be supported by endogenous ferredoxin and ferredoxin reductase [ 17 , 18 ]. it appears that endogenous redox partners are also present in sufficient quantity to allow the high - level of expressed cyp to be functional within the periplasm without the need for coexpression or addition of the native counterparts . this validated the approach as a suitable one in order to obtain the highest yield of heterologous cyp1a1 in the literature coupled with an active protein in an advantageous accessible cellular location . in the present study several interesting and novel features of e . coli periplasmic space as an ideal location for targeting recombinant , human cyp ( s ) have emerged . previously , the heterologous expression of human cytochromes p450 have employed many systems including bacterial [ 19 ], yeast [ 20 ], and cos cells [ 21 ]. the normal methods for bacterial expression of membrane - bound mammalian cytochromes p450 , as pioneered by barnes et al ., [ 19 ], involved the modification of the initial coding triplets . subsequently , several groups have examined expression of human cytochromes p450 using the pelb and ompa leader sequences together with the tac promoter [ 6 , 7 ]. however , their experiments did not produce removal of the leader sequence such that non - authentic recombinant p450 was formed . furthermore , movement of the cytochrome p450 into the periplasmic space was not demonstrated and hemoprotein yields were typical of previous studies producing a few hundreds munol cyp / l bacterial culture at best . recently , cytosolic expression has been reported in the 2 - 3000 nmol / l range by incubating cells in the presence of subtoxic levels of chloramphenicol [ 22 ], but not to the 4500 nmol / l observed here using the phoa promoter . the periplasm is a more oxidising environment than the cytosol , but results here show it provides a stable environment for accumulation of significant amounts of correctly folded and heme - associated cyp . the reason for the significantly high level of production of functional cyp may lie in the present strategy of targeting the protein to the periplasmic space , where the appropriate post - translational enzymic machinery is localised for heme incorporation . 5 - aminolevulinic acid is the fist committed precursor in e . coli tetrapyrrole biosynthesis [ 23 ]. it can be derived from the c - 5 ( glutamate ) pathway , more commonly , and a route involving the condensation of succinyl coa and glycine ( 24 ]. biosynthesis from 5 - aminolevulinic acid to iron protoporphyrin dc ( protoheme heme b ) occurs in the cytosol involving the proteins coded for by hemb - g . in a subsequent step , formation of heme - thiolate proteins such as cyp requires insertion of the protoheme into the pocket of the apoprotein together with the formation of the fifth axial thiolate ligand of the cyp cysteine residue . information on how this occurs is poorly understood . as e . coli does not produce cyp it would appear protoheme incorporation may occur spontaneously or autologously . cytochrome of the c type , containing covalently bound heme c are widely distributed in nature where they function in photosynthetic and respiratory electron transfer chains [ 25 ]. e . coli does not contain a cytochrome bc 1 complex analogous to the mitochondrial electron transport chains components , but does contain a related cytochrome , cytochrome c 552 , that contains six covalently bound heme groups and is located in the periplasm functioning as dissimilatory nitrate reductase [ 26 ]. the heme required for cyp production may be produced as for other cytochromes in this cellular compartment . in e . coli no heme lyase activity or related coding gene are known and it is thought that cytochrome c assembly occurs spontaneously . recently , cydc and cydd were identified , which are required for biogenesis of c - type cytochrome [ 27 ]. either gene disruption prevented synthesis of cytochrome bd quinol oxidase . both cydc and cydd are required for cytochrome c assembly and are also involved in heme transport to the periplasmic space where both types of cytochrome appear to be assembled . dsb proteins also appear to be important in maintaining the thiol groups of the periplasmic apopoteins in a suitably reduced state . again it remains to be seen whether formation of thiolate moiety of the cyp apoprotein requires such participation of the dsb chaperones for assembly of cyp in the periplasm of e . coli [ 28 ]. we show here significant in vivo drug transformation capability of recombinant native cyp1a1 within e . coli . this cyp is a typical microsomal enzyme in eukaryotes , relying on nadph - cytochrome p450 reductase for activity . here an nadph - dependent activity was found . surprisingly , the periplasm of e . coli is aptly furnished with endogenous electron donors for in vivo transformation capability of the targeted cyp1a1 . consequently , the periplasmic location bypasses the permeability barrier of the inner membrane and thus promises wider applications of this heterologous p450 expression system for diverse industrial applications such as drug metabolite production for toxicological screening . alternatively , it may be useful for bioremediation of environmental pollutants as cyp1a1 includes amongst its substmtes many recalcitrant polycyclic aromatic hydrocarbons [ 29 , 30 ]. we also conclude that the periplasrn of e . coli is an ideal compartment for targeting recombinant human cyp ( s ). this may be considered a preferential route to allow a high yield of microsomal cyps , which may be useful in structural studies on this important superfamily of proteins . 2 export of cytochrome p450105d1 to the periplasmic space of escherichia coli in this example , using methods generally as set out in example 3 . streptomyces griseus cyp105d1 was appended at its amino terminus to the secretory signal of escherichia coli alkaline phosphatase . the chimeric p450 construct was placed under the transcriptional control of the nativephoa promoter in a prokaryotic expression vector . induction of the hemoprotein by heterologous expression in e . coli following growth in a phosphate - limited medium resulted in abundant synthesis of recombinant cyp105d1 as detected by reduced co - difference spectra . the signal - appended cyp105d1 was translocated across the bacterial inner membrane by a sec - dependent pathway and processed to yield authentic , heme - incorporated p450 within the periplasmic space . in vitro and whole cell metabolic activity studies showed that the periplasmically - located cyp105d1 competently catalysed nadh - dependent oxidation of the xenobiotic compounds benzo [ a ] pyrene and erythromycin , further revealing the presence in the e . coli periplasm of endogenous functional redox partners . cyp10sd1 has previously been expressed as a recombinant cytosolic form in escherichia coli using the iptg inducble lac promoter and enhanced drug transformation activities were observed in the enzyme preparations from thee bacterial cell lysates ( 32 ). due to selective permeability of e . coli to many substrates / products , serious problems can arise in the optimisation of biotransformiation processes using whole cell systems . for such reasons , cell wall mutants of salmonella typhimurium were developed for use in mutagenesis tests ( 19 ). one approach to overcome these problems would be to engineer cytochromes p450 that can be exported to the periplasm or the cell exterior . in this experiment we engineered a chimeric secretory form of s . griseus cytochrome p450 ( cyp105d1 ) that , when expressed in e . coli , leads to abundant synthesis of the precursor protein . the precursor was exported via the sec - dependent pathway to the periplasm where it was correctly processed and incorporated heme to yield a functional hemiprotein . we also show that periplasm of e . coli has the necessary endogenous redox partners to racilitte in vivo biotransformations which can have value for biocatalysisl bioremediation strategies the e . coli strains employed were those described in example 3 and the bacteria were propagated and harvested under conditions substantially as set out in example 3 . in this example , the dh5a strain was used as a foster strain for introduction of in vitro ligated plasmid dna whereas tb1 strain was subsequently employed for expression of the recombinant cypi05d1 . the cyp105d1 open - reading frame , cloned in the expression vector pliq , was induced by growth of e . coli in phosphate - limited ( 0 . 1 mm ) mops medium ( 30 ) in the presence of 100 μg ampicillin / ml at 30 ° c . for specified periods ; a saturated lb - culture was used as a 2 % ( v / v ) starter inoculum . these were carried out according to the general methods set out in example 3 . genomic dna , isolated from a 100 ml culture of streptonyces griseus as described previously by trower et al . ( 34 ), was used as the template to amplify cyp105d1 . the gene was amplified as a pcr fragment of 1239 bp containing the engineered hindiii and psti sites at 5 ′ and 3 ′ ends , respectively , using the following forward and reverse primers : the pcr conditions were as described above . the resulting cyp105d1 dna fragment was doubly digested with hindiii and psti and ligated into the previous hindiii and psti - cut pliq e . coli expression plasmid . the restriction and dna modifying enzymes were purchased from promega ( southampton ) and their use followed as recommended by the supplier . [ 0087 ] e . coli cubivation , harvesting and subcellular fiactionations were carried out as kn example 3 . cyp content in biological samples were monitored by oxidised versus reduced difference spectroscopy of co - bound hemoproteins according to omura and sato ( 25 ) using the molar absorption coefficient of 91000 m − 1 cm − 1 . extracts containing p450 were diluted with 100 mm potassium phosphate buffer ( nh 7 . 4 ) containing 1 mm na 2 edta and 20 % ( v / v ) glycerol and light - absorption spectra measured using a hitatehi u3010 scanning spectrophotometer . cyp105d1 enzyme activities were monitored in a 1 ml final reaction volume composed of 200 pmol of recombinant cyp105d1 ( contained in an appropriate volume of e . coli periplasmic fraction or cells ). the reaction was initiated by addition of nadh ( 1 mm final concentration ). for assessing enzyme activity in whole cells of induced e . coli tb1 harbouring either the control ( pliq ) or recombinant plasmid ( plicyp105 ) following , the harvested cells ( 5000 g × 2 min ) were washed twice with 0 . 1m potassium phosphate buffer ( ph 7 . 4 ) containing 20 % ( v / v ) glycerol and finally resuspended in 1 ml of the same buffer for incubation with substrate . erythromycin n - demethylation activity was determined as described previously ( 4 ). benzo [ a ] pyrene 3 - hydroxylation activity was measured fluoroinetrically using a perkin elmer fluorescence spectrphotometer according to the method of nebert and gelboin ( 22 ). the protein content in bacterial fractions was estimated using the bicinchoninic acid ( signa chemicals ) assay using bovine serum albumin as standard . protein patterns were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) employingthe discontinuous buffer system of laemmli ( 13 ); approximately 75 μg of protein was loaded per lane . this example describes the development of an expression system for efficient targeting of cytochrome p450 to the periplasmic space of e . coli stain tb1 using the plasmid pliq . this expression vector is a derivative of the previously described paa - cyt ( 11 ), re - engineered with appropriate restriction sites downstream of the alkaline phosphatase signal sequence . previous studies for the successful expression of cytochromes p450 in e . coli have required modification at the 5 ′ end of the p450 gene or necessitated installation of a leader sequence ( peib ) 5 ′ to the p450 cdna ( 3 , 27 ). in the present study , the gyp 105d1 cdna was directly fused with the signal sequence and placed under the tight transcriptional control of the phoa promoter , inducible by growth of the bacteria in a phosphate - limited medium ( 30 ). whole lysates derived from e . coli induced for 20 h to express protein displayed the characteristic p450 co - difference absorption spectra with defined spectral maxima of the soret absorption at 448 nm that is a distinctive hallmark for cytochrome p450s ( fig2 ). the progressive increase in the soret absorbance peak with culture growth accompanied the intensifying red colour of the bacteria that was visually recoverable in the periplasmic fractions . this indicated derivation from de novo synthesis of a cyp - related hemoprotein . interestingly , no significant peaks at around 420 nm ( an inactive form p450 ) in the freshly prepared cell - free extracts were observed prior to peak induction periods . this is in contrast to that reported for the production of the recombinant cytoplasmic counterpart ( 32 ) and suggested stable and functional production of cyp105d1 . a time - course profile of the production of cyp105d1 in the isolated periplasmic fractions was undertaken by estimating the hemoprotein content by difference spectroscopy ( fig3 ). this study revealed that detectable periplasmic build - up of cyp10sd1 occurs around 8 h and steeply rises to a peak point around 25 h from the start of growth . following this period the levels of the cytochrome rapidly decline . the extent of the cyp105d1 production is indeed dramatic reaching a peak value exceeding 600 nmoles per litre of culture following addition of δ - aminolevulinic acid as a heme precursor . in some experiments & gt ; 1000 nmol of cyp105d1 have been obtained from this expression system . this level of hemoprotein is well in excess of the published figures for e . coli expression of cytochrome p450 in general . previous studies reporting teheterologous expression of cyp105d1 under the control of the strongly inducible tac promoter obtained levels of cyp105d1 hemoprotein just over 400 nmol of p450 recovered in cytosol / litre culture when e . coli was cultured in the presence of 1 mm δ - ala ( 32 ). the present results indicated that heme could be successfully incorporated into p450 during folding of matured protein trammiocated into the periplasmic space of e . coli . in order to further investigate the subcellular localisation of the recombinant cyp , the bacteria induced for expression of the protein for 20 h were subfractionated into periplasmic , cytoplasmic and membrane fractions . that effective subcellular fractionation had occurred was noted by & gt ; 90 % enrichment of the marker enzyme activity associated with the isolated subcellular fraction : alkaline phosphatase ( periplasm ), malate dehydrogenase ( membranes ) and furiarase ( cytosol ). more man 80 % of the total cellular cyp105d1 content was found localised in the periplasmic space of e . coli . further substantiation of the cellular location of cyp forms were sought by subjecting the periplasmic protein fractions derived from e . coli tb1 expressing cyp105d1 to sds polyacrylamide gel electrophoresis . the results show induction of a novel protein of the expected size of 45 , 000 d . moreover , the polypeptide profile of the periplasmic fraction derived from e . coli expressing cyp105d1 show dramatic changes in the overall polypeptide composition when compared with counterpart control harbouring a plasmid without cyp105d1 gene . the most significant change is the intense co - overproduction of a 30 kda protein whose identity was confirmed as β - lactamase through n - terminal protein sequence of the gel isolated protein band . a periplasmic fraction derived from e . coli plicyp105d1 following extensive dialysis against water was subjected to electron spray analysis . molecular weights parsed around the expected region of cyp105d1 deconvoluted a major protein species with a molecular weight of 45406 da , 4 da lower than the size determined from the sequence of the cloned gene placed downstream of the secretory sequence . this verified that the exported protein was correctly processed by signal peptidase following its translocation across the inner membrane of e . coli . periplasmically targeted cyp105d1 is enzymically active in in vivo and ex vivo the catalytic activities of recombinant cyp105d1 was measured in vivo using whole cell biotransformation procedure and in vitro with isolated periplasmic fractions expressing cyp105d 1 . no activity was recorded in control samples derived from e . coli tb1 harbouring the empty plasmid pliq . the data obtained demonstrated that the activity of periplasmic targeted cyp105d1 was comparable to previous reports for such activities ( 32 ). the ability of the isolated fraction to sustain the xenobiotic transformation in e . coli periplasmic fraction strongly suggests that this compartment contains suitable electron donor proteins for p450 in the perplasmic space of e coli ; the native cyp105d1 requires a ferredoxin and ferredoxin reductase . it appears that the foreign redox partners are also present in sufficient quantity to allow the high - level of expressed protein to be functional without the need for co - expression or addition of the native counterparts . this validated the approach as a suitable one in order to obtain high yields of heterologous cyp105d1 coupled with an active protein in a novel cellular location . the heterologous expression of cytochromes p450 have employed many systems including bacterial ( 2 ), yeast ( 24 ), baculovirus ( 1 ) and cos cells ( 35 ). reasons supporting this interest are described in the introduction and include involvement of cyp in current and potential industrial biotransformations as well as production and characterisation of proteins central to the toxicological fate of organic xenobiotics . the normal method for bacterial expression of membrane - bound eukaryotic cytochromes p450 involves the extensive modification of the n - terminus , although the soluble cyplosdi did not require this for expression in the cytosol ( 32 ). expression of cytochromes p450 using the pelb and ompa leader sequences have been examined together with the tac promoter ( 3 , 27 ). these previous studies did not reveal removal of the leader sequence or demonstrate movement of the cytochrome p450 into the periplasm and yields were typical of previous studies producing a few hundred mnol cyp / l culture . recently , cytosolic expression of cyp 17 has been reported in the range from 2000 to 3000 nmol p450 / lit culture by incubation of cells in the presence of subtoxic levels of chloranphenicol ( 20 ). we also saw similar levels in the system described here on some occasions . in this study several interesting and novel features of e . coli periplasmic space as an ideal site for targeting recombinant cyp have emerged which merit further discussion . firstly the periplasm although a more oxidising environment than the cytosol provides a stable environment for accumulation of significant amounts of correctly folded and heme - associated holocyp over extended periods of cultivation . secondly , drug transformation capability of native cyp105d1 is intrinsically dependent upon two additional redox partners , namely a ferredoxin and the cognate ferredoxin reductase . surprisingly , the periplasm of e . coli is aptly furnished with the endogenous electron donors for in vivo transformation capability of the targeted cyp105d1 . this together with broad substrate range of cyp105d1 and a location that bypasses the permeability barrier of the inner membrane indicates a potential for wider applications of the heterolous expression system for diverse industrial applications . the reason for the significantly high level of production of functional holocyp may lie in the present strategy oftargeting the protein to the periplasm where the appropriate post - translational enzymic machinery is localised for heme assembly . in e . coli 5 - aminolevulinic is the first committed precursor in tetrapyrrole biosynthesis ( 18 ). there are two routes for the derivation of 5 - aminolevulinic acid , the c - 5 ( glutamate ) pathway , which is now thought to be more common in the biosphere , and a route involving the condensation of succinyl coa and glycine ( 6 ). the complex steps in biosynthesis from 5 - aminolevulinic acid to iron protoporphyrin ix ( protoheme = heme b ) occurs in the cytosol involving a battery of enzymes coded for by hemb - g . in a subsequent step , formation of hem - thiolate proteins such as cyp require insertion of the protoheme into the pocket of apoprotein together with fifth axial bridging via thiolate of cysteine residue . since e . coli is not naturally endowed with synthesis of a cyp , or for that matter related heme - thiolate proteins , it would appear protoheme incorporation must occur spontaneously or autologously . cytochrome of c type , which contain covalently bound home c ( derived by ligation of the vinyl groups to cysteine residues on the apoprotein ) are widely distributed in nature where they function in photosynthetic and respiratory electron transfer chains ( 20 ). although e . coli is capable of aerobic respiration it lacks soluble or a cytochrome bc 1 complex analogous to the mitochondrial electron transport chains components . the major related cytochrome c in e . coli is cytochrome c 552 that contains six covalently bound heme groups and located in the periplasm , where it functions as dissimilatory nitrate reductase ( 10 ). in contrast to eukaryotes , no heme lyase activity or related coding gene has been identified in e . coli and so it so it is thought that cytochrome c assembly occurs spontaneously . recently two e . coli genes cydc and cydd whose products are specifically required for biogenesis of c - type cytochrome have been identified ( 26 ). disruption of either of these genes prevents the synthesis of cytochrome bd quinol oxidase . the cydc and cydd are proteins not only required for cytochrome c assembly but are also involved in heme transport to the periplasmic space where both types of cytochrome appear to be assembled . more recent findings implicate the role of dsb proteins in maintaining the thiol groups of the periplsmic apopoteins in a suitably reduced state for assembly with heme b prosthetic group ( 21 ). it remains to be seen whether thiolate moiety of the apoprotein requiring the fifth axial co - ordination with the heme b also requires such participation of the bsb chaperones in the assembly of cyp in the perplasm of e . coli . we therefore conclude that the periplasm of e . coli an favourable compartment for targeting recombinant cytochrome p450 . 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