Abstract:
A method of protein synthesis which comprises providing a genetic unit comprising a nucleotide sequence coding for a pre-form apo-protein. The pre-form apo-protein is synthesized in a cytoplasmic region of a cell and translocated to the periplasmic region of the cell for conversion to a corresponding holo-protein.

Description:
FIELD OF THE INVENTION 
     The present invention is concerned with protein synthesis and in particular with the production of recombinant fusions between cytochrome b 5  and foreign proteins which can be translocated out of the cell into the periplasmic space of, for example, Escherichia coli. 
     BACKGROUND OF THE INVENTION 
     The mechanism by which a cytoplasmically-synthesised (recombinant) protein is translocated into the periplasmic space of Escherichia coli is known to occur by the workings of the signal hypothesis (see review by Stardler, J. A. and Silhavy, T. J., 1990, Methods in Enzymology, 167-187). Many such proteins are initially synthesised as precursor forms carrying extensions at their amino termini known as signal sequences (peptides). The signal sequence has the information required for selective translocation of the passenger part of the protein molecule across the cytoplasmic membranes of the bacteria. The signal is cleaved off soon after the periplasmically-deposited protein has gained its native biological fold and function. 
     SUMMARY OF THE INVENTION 
     Using this approach we have developed a bacterial system which allows production of chimeric or fusion forms of coloured proteins. These proteins are secreted into the periplasm where they are afforded greater protection against potential degradation. 
     According to a first aspect of the present invention there is provided a method of protein synthesis which comprises provision of a genetic unit comprising a nucleotide sequence coding for a pre-form of an apo-protein, synthesizing said pre-form apo-protein in a cytoplasmic region of a cell, and translocating said synthesised pre-form apo-protein to the periplasmic region of said cell, so as to permit constitution in said periplasmic region of a signal-processed apo-protein which can then be converted to the corresponding holo-protein. 
     According to a second aspect of the present invention, there is provided a genetic precursor unit which comprises a nucleotide sequence coding for a pre-form apo-protein, said nucleotide sequence being such that said pre-form apo-protein is translocatable from a cytoplasmic region of a cell to the periplasmic region of said cell, so as to permit constitution in said periplasmic region of a processed apo-protein and conversion into a corresponding holo-protein. 
     The genetic unit is preferably suitable for expression in the cytoplasmic region of a bacterial host cell, such as an E.coli host cell. The preferred E.coli strain is TB-1,  F ara Δ (lac-proAB) rps φ 80d lacZΔM15 hsdR17 (rk+ m   +  k)! or N4830-1  f-suo his-ilv galK-(λch1D-pg1)(λBam N+CI857 H1)!. 
     A preferred apo-protein comprises a cytoplasmic cytochrome, which typically comprises the soluble core domain of cytochrome b 5  of liver endoplasmic reticulum. 
     Cytochrome b 5  of the endoplasmic reticulum of mammalian liver is a well characterised, small haemoprotein of 16.7 kD which plays a central role in a variety of electron transfer reactions related to fatty acid desaturation, redox cycling of oestrogen and reduction of cytochrome P-450 reductase. 
     The hepatic cytochrome b 5  is composed of two domains, the above-described soluble, enzymatically active, haem-containing globular core (b 5 ) of about 12 kD and a smaller carboxy terminal tail anchored in the microsomal membrane. Within the tail portion, a stretch of 23 hydrophobic amino acid residues, also definable as an &#34;insertion&#34; sequence, autonomously and post-translationally integrates cytochrome b 5  into the lipid bilayer of the endoplasmic reticulum such that the active core domain is laterally disposed from the reticulum facing the cytoplasm. 
     It is preferred that the genetic unit further comprises a nucleotide sequence which codes for an amino-terminal signal peptide (the pre region of pre-form apo-protein) which is recognised by the cell to direct the pre-form apo-protein to the cytoplasmic membrane and thence subsequently translocated into the periplasm of the cell. The signal peptide typically comprises E.coli alkaline phosphatase, which is advantageously in linkage with cytochrome b 5  (apo-protein). The method therefore preferably further comprises the step of directing the apo-protein to the cytoplasmic membrane, under the direction of the signal peptide, so as to facilitate the above-described translocation of the pre-form apo-protein from the cytoplasm to the periplasmic space of the bacterium. 
     In this preferred embodiment of the present invention where the genetic unit further comprises a nucleotide sequence which codes for an amino-terminal signal peptide, the invention can be used to investigate signal peptidase which in vivo proteolytically cleaves N-terminal signal peptides. 
     Many proteins which are synthesised in the cytosol have to be targeted to various extracellular and subcellular locations. This involves translocation across one or more lipid bilayers impermeable to large hydrophilic molecules. A feature of most translocated proteins is that they are synthesised as precursor forms carrying N-terminal signal sequences, which as discussed above are proteolytically cleaved by signal peptidase, either during or immediately after transport of the passenger protein across the biological membrane. 
     Signal peptidase is highly specific as it cleaves only preprotein precursors. Although lacking sequence homologies, signal sequences are functionally highly conserved. Despite significant variations in their lengths, all signals have three definable regions, namely an N-terminal region carrying one or two basic residues, a middle core encompassing hydrophobic residues and a terminal region specifying the cleavage site. 
     The conventional assay for signal peptidase has until now relied on the use of natural precursor proteins which are synthesised in a cell-free translation system in a highly radioactive form. The extent of processing is monitored by incubating the radio-labelled substrate with either membranes or signal peptidase and monitoring the processed bands by autoradiography following gel electrophoresis. The assay is not only time-consuming and expensive but suffers from the use of minuscule quantities (fmol) of substrates in relation to enzyme quantities, with inevitable contamination by many cellular components derived from the use of in vitro synthesis systems. 
     We have now overcome the above problems, and there is further provided by the present invention an assay system for signal peptidase, which assay system comprises: 
     (a) a genetic precursor unit which comprises a nucleotide sequence coding for an apo-protein and an amino-terminal signal peptide, the nucleotide sequence being such that the apo-protein is translocatable from a cytoplasmic region of a cell to the periplasmic region of the cell, so as to permit constitution in said periplasmic region of a processed apo-protein; and 
     (b) signal peptidase, a source thereof or a precursor therefor, wherein the signal peptidase can effect proteolytic cleavage of the amino terminal signal peptide from the apo-protein. 
     The genetic unit of the assay system is substantially as hereinbefore described and is advantageously suitable for expression in a bacterial medium such as E.coli and the assay system preferably further comprises a host medium such as E.coli substantially as hereinbefore described. 
     Preferably the assay system further comprises means for monitoring the proteolytic cleavage of the amino terminal signal peptide from the apo-protein. The monitoring means typically comprises means for separating the cleaved apo-protein from the pre-form apo-protein, and means for spectrophotometrically monitoring the cleaved apo-protein following supplementation thereof with exogenous haem. Typically the pre-form apo-protein is phase-separated from the cleaved apo-protein, the pre-form only being soluble as an aggregate in the presence of a detergent, the cleaved apo-protein being water soluble. 
     The provision of such an assay system is advantageous in investigating the mechanistic and structural properties of signal peptidase. A particularly useful application of an assay system according to the present invention is in the identification of inhibitors of signal peptidase which can subsequently be used as probes in investigating the reaction mechanism of signal peptidase. 
     There is further provided by the present invention a method of identifying inhibitors to signal peptidase, which method comprises providing an assay system substantially as described above, introducing a test material into cells of a host medium used in the assay system, and monitoring the effect of said test material on a selected activity of the signal peptidase. 
     Advantageously, the identification method involves monitoring the cleavage of the amino-terminal signal peptide from the apo-protein by spectrophotometric means substantially as hereinbefore described. 
     Preferably the test material comprises any of the following: a consensus signal peptide (which typically comprises a 19 residue peptide), one or more transition metal ions, and carboxy-modifying reagents. 
     Signal peptidase is a known crucial rate limiting step in the pathway of protein secretion and can offer a locus for control or regulation of an essential biofunction, namely protein export. Thus the identification of a signal peptidase inhibitor can provide a tool to understand the reaction mechanism of signal peptidase, and also, more importantly, a means to regulate growth of microorganisms and protein secretory disorders in higher organisms. 
     The invention allows the observation of the effects of competitive inhibition by selected inhibitors in order to identify the precise region of signal sequence that is essential for recognition/cleavage by signal peptidase. 
     There is further provided by the present invention a method of recognising an active binding site of signal peptidase, which method comprises contacting the signal peptidase with at least on photoactivatable form of a radio-labelled (typically radioiodinated) inhibitor of the peptidase, and identifying the binding location of the inhibitor to the peptidase. 
     There is further provided by the present invention, a test material identified as an inhibitor to signal peptidase, for use in modulating in vivo protein secretion. 
     The test material may advantageously comprise a synthetic inhibitor of signal peptidase, and has beneficial applications as a biocidal agent, regulating secretory disorders in mammalian cells, control of plant growth and the like. 
     The genetic unit typically comprises a plasmid having a vector as shown in any of FIGS. 1 to 3, or a DNA sequence as shown in any of FIGS. 4 to 6 (SEQ ID NOS 1 to 3, respectively). The most significant features of the plasmid sequences are as follows: 
     a) a native pho promoter for transcriptional control. The pho promoter is available coded for by the plasmids pSEC-cyt which is inducible by growth of the bacteria in a phosphate-limited medium; 
     b) a signal sequence coding for alkaline phosphatase as hereinbefore described; and 
     c) a soluble cytochrome b 5  core gene sequence. 
     There is further provided by the present invention a genetic unit having a vector substantially as illustrated in any of FIGS. 1 to 3, or a DNA sequence as illustrated in any of FIGS. 4 to 6 (SEQ ID NOS 1 to 3 respectively), which is capable of generating translocated forms non-fusion (FIG. 1 or 4) or fusion (chimeric) forms of coloured cytochrome b 5  (FIGS. 2,5 and 3,6). The foreign protein can be tagged to the cytochrome b 5  protein at its carboxy (C) FIGS. 2,5) or amino (N) terminus (FIGS. 3,6). 
     The pho promoter is typically capable of minimising the lethality of the expressed chimera to the host by maintaining the expression of chimera under the tight transcriptional control of the native pho promoter, in plasmids pSEC-cyt/C and pSEC-cyt/N, inducible only by growth of the bacteria in a phosphate limited medium. In pSEC-cyt/C fusions are introduced by linkage of a foreign DNA sequence at the carboxy terminus of cytochrome b 5  gene through the unique BclI site. In pSEC-cyt/N fusions are introduced immediately at the N-terminus of cytochrome b 5  but after the signal sequence, through the unique BamHI and/or PstI sites. 
     The method of expression of the pre-form cytochrome b 5  or its chimeric derivative typically involves initiation of transcription, and hence protein synthesis, by control of the incubation conditions of the host medium. Typically the initiation stage comprises cultivation of the bacterial host in a phosphate-limited medium (0.1 mM phosphate), and spectrophotometric or visual monitoring (transformation of the colour of the bacterial cells from a grey to pink colour) of the synthesis cytochrome b 5  (processed) or its chimera (processed) in E.coli harbouring pAA-cyt or pSEC-cyt, respectively, under the transcriptional control of the pho promoter. 
     It is further preferred that the method involves translocation of a prosthetic group from the cytoplasmic region of the cell to the periplasmic region, for combination with the signal-processed apo-form of the chimeric protein in the periplasmic region to form the holo-protein as hereinbefore described. It is preferred that the apo-protein is first translocated to the periplasmic region, followed by subsequent translocation of the prosthetic group, such that the apo-protein can act as a sink for the latter. In the case where the apo-protein comprises the cytochrome b 5  soluble core, the prosthetic material comprises the haem (protoporphyrin IX) prosthetic group. The periplasmic accrual of the apo cytochrome b 5  core is thought to trigger the subsequent translocation of the haem-prosthetic group. 
     It is envisaged that a prosthetic material may be present in the periplasmic region of the cell, although this does not appear to be the case with cytochrome b 5  due to the observed build up of periplasmic apo-cytochrome b 5  and ensuing time lag preceding the appearance of periplasmic holo-cytochrome b 5 . 
     Production of the exported periplasmically-localised haemoprotein can typically exceed 6 mg/liter of culture after five hours induction. Mutated forms of the cytochrome b 5  have been developed which can enhance exportation of the mature forms by more than tenfold. The approach may be useful for the production and secretion of coloured forms and fusions in eukaryotic cells. 
     The method according to the invention typically further comprises monitoring of protein synthesis by observing the visual transformation of the bacterial cells from a grey translucent to a bright pink colour. This colour change is due to the expression of the cytochrome b 5  gene. The monitoring may be carried out by spectrophotometric means where an accurate analysis of the extent of protein synthesis is required, so allowing the protein synthesis to be terminated when sufficient protein has been synthesised. 
     The above-described method allows a prokaryotic signal sequence to be appended with cytochrome b 5 , such that this natural cytoplasmic protein of eukaryotic origin is transformed into a secretory form in E.coli. Furthermore, the expression of the eukaryotic gene encoding the pre-form apo-protein or its chimera, synthesised in the cytoplasm, is such that the post-translocationally processed component in the periplasm gives rise to the holo-protein or its fusion counterpart. 
     According to the present invention there is further provided a proteinaceous material comprising a pre-form apo-protein which is synthesised in the cytoplasmic region of a cell, the pre-form apo-protein being translocatable to the periplasmic region of said cell, so as to allow constitution in said periplasmic region of a correspondingly processed apo-protein, the binding of a prosthetic group (typically the haem group) to the apo-protein forms the holo-protein. 
     There is further provided by the present invention an apo-protein substantially as hereinbefore described, in combination with a signal peptide for selective translocation (export) or secretion of said apo-protein from the cytoplasmic to the periplasmic region of a cell, so as to allow constitution in the periplasmic region of a corresponding holo-protein from said apo-protein. 
     According to yet a further aspect of the present invention there is provided a holo-protein constituted in the periplasmic region of a cell from an apo-protein, or by a method, substantially as hereinbefore described. There is further provided a holo-protein derived from a genetic precursor unit, substantially as illustrated with reference to any of FIGS. 4 to 6. 
     The above-described apo- and holo-proteins typically respectively comprise the apo- and holo- forms of cytochrome b 5  or its respective N- or C-terminally fused chimeric forms. The fusions can be generated at either the amino or carboxy terminus of the secreted recombinant protein. 
     There is further provided by the present invention an E.coli host medium containing a genetic precursor unit substantially as hereinbefore described. 
     There is further provided a kit for use in protein synthesis, which kit comprises: 
     a) a bacterial host medium substantially as hereinbefore described; and 
     b) genetic material comprising a nucleotide sequence coding for a pre-form apo-protein, such that said pre-form apo-protein coded for by said nucleotide sequence is translocatable from the cytoplasmic to the periplasmic region of a cell of the host medium, so as to allow constitution in the periplasmic region of a holo-protein from the apo-protein. 
     The kit typically comprises a bacterial host medium, such as E.coli, together with genetic material comprising precursor constituent genes suitable to be included in a genetic unit substantially as hereinbefore described. 
     An example of a typical synthesis of a fusion protein according to the present invention will now be described. 
     A gene coding for a foreign protein or peptide was fused in a reading frame from either (i) the carboxy-terminus or (ii) the N-terminus of the soluble core of cytochrome b 5 . The foreign gene was ligated between either (i) the unique BclI restriction site and the downstream polylinker site or (ii) the unique PstI and BamHI site located between the signal sequence and cytochrome b 5 . 
     E.coli TB-1 (Lac) cells were then transformed with the ligation mixture. 
     Transformants were plated out on Luria broth agar (1.5% (w.v)) plates containing ampicillin (75 μg/ml), isopropyl-β-D-galactopyranoside (IPTG) (100 μM),5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (20 μg/ml). Transformant colonies bearing potential inserts were white in colour whereas non-recombinants were blue in colour. If required, the recombinant plasmids could be further verified by restriction mapping. 
     For induction, a starter bacterial culture cultivated to saturation in LB/ampicillin was applied at a 2% (v/v) inoculum into MOPS medium. The cultures were grown with shaking at 100 revs/min at 35° C. for 5 to 10 h. 
     The following table indicates the composition of the MOPS medium. All solutions were sterilised by autoclaving or filter sterilisation. The final concentration of phosphate in the MOPS medium was about 0.1 mM. 
     
         ______________________________________Component       % (v/v) in MOPS______________________________________Water           74.5M.sup.a         20O.sup.f         0.2P.sup.a         0.01S.sup.a         0.1VFCAA.sup.a     220% glucose.sup.f           10.05% thiamine B1.sup.f           0.225 mg/ml ampicillin.sup.f           0.3______________________________________ M, 4.2% (w/v) MOPS buffer, 0.4% (w/v) Tricine, 1.46% NaCl, 0.8% KOH, 0.2555% NH.sub.4 Cl O, 5.36% MgCl.sub.2, 0.16% HCl, 0.1% FeCl.sub.2, 0.00368% CaCl.sub.2.2H.sub.2 O, 0.00128% H.sub.3 BO.sub.3, 0.0008% MnCl.sub.2.4H.sub.2 O, 0.00036% CoCl.sub.2.H.sub.2 O, 0.00008% CuCl.sub.2.2H.sub.2 O, 0.0068% ZnCl.sub.2, 0.0121% Na.sub.2 MoO.sub.4.2H.sub.2 O. P, 1.0M KH.sub.2 PO.sub.4 S, 0.276M K.sub.2 SO.sub.4 VFCAA, 7.5% vitaminfree Casamino acids .sup.a autoclaved prior to use .sup.f filter sterilised prior to use 
    
     Positive expression of the fusion protein was indicated by pink colour of the bacterial cells obtained by centrifugation at 5,000×g for 5 min. 
     The protein was extracted from the bacteria by the following &#34;osmotic shock&#34; method: 
     (i) The cells (derived from a 30 ml culture) were gently suspended in 1 ml of 20% sucrose, 0.3 M Tris-HCl (pH 8), 1 mM Na 2  EDTA)(STE) and incubated at room temperature (22° C.) for 10 min. 
     (ii) The cells were then harvested as pellet by centrifugation at 5,000×g for 6 min and thoroughly resuspended in residual STE volume. 
     (iii) The plasmolysed cells were osmotically shocked by rapidly suspending in ice-cold 0.5 mM MgCl 2  and left on ice for 10 min. 
     Centrifugation at 10,000×g for 10 min yielded supernatant comprising the periplasmic fraction. Pink coloration of the supernatant implied that fusion protein had been efficiently extracted from the periplasmic space. 
     The quantity of the recombinant protein was monitored by subjecting a fraction of the extract to a spectral scan from 350 to 500 nm. The oxidised cytochrome was identifiable by the rise in absorbance at 413 nm. Often a significant pool of the apo-protein may be present. This may be converted to holo cytochrome by addition of haem to the supernatant to a final concentration ranging from 5 to 20 μM; an equivalent amount of haem was added to the blank cuvette to counteract the absorbance rise by excess haem in the sample. The amount of protein may be calculated from the mM extinction coefficient of the oxidised cytochrome b 5  of 115 and 413 nm. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The present invention will now be further illustrated, by way of example only, by reference to the accompanying FIGS. 1 to 6, wherein: 
     FIGS. 1,2 and 3 illustrate expression vectors pAA-cyt, pSEC-cyt/C and PSEC-cyt/N of a genetic unit according to the present invention; 
     FIGS. 4-6 illustrate the genetic sequences of the vectors respectively illustrated in FIGS. 1-3 (SEQ ID NOS 1 to 3, respectively); and 
     FIGS. 7-9 schematically illustrate modifications of plasmids according to the present invention, which involve cloning, expression and isolation of the chimeric proteinaceous material. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 1 illustrates plasmid PAA-cyt constructed for the periplasmic production of non-fusion, secreted holo-cytochrome b 5 . 
     FIG. 2 illustrates the plasmid PSEC-cyt/C constructed for linkage at the 3&#39; terminus (C-terminus) of the soluble cytochrome b 5  core gene with a foreign gene sequence, according to the present invention. 
     FIG. 3 illustrates the plasmid PSEC-cyt/N for linkage at 5&#39; terminus, between the C-terminus of alkaline phosphatase signal and the N-terminus of cytochrome b 5  of the cytochrome b 5  core gene, according to the present invention. 
     Referring now to FIGS. 1 to 3, the essential features of the illustrated vector are as follows: 
     Pho, Pho promoter activated in bacteria grown on phosphate limited medium; 
     S/D), Shine Dalgarno sequence for translation initiation of alkaline phosphatase signal; 
     SS, alkaline phosphatase signal sequence gene encoding 21 residue signal including the first arginine residue of mature alkaline phosphatase; 
     Cyt, soluble core of cytochrome b 5  ; 
     T, tail of the native cytochrome b 5  ; 
     Lac, Lactose operon promoter; and 
     PLS, polylinker sites: EcoRI, (Hind III), SacI, KpnI, SmaI, BamHI, XbaI, SalI, HincII, BspMI, PstI, SphI, HindIII. (In the polylinker sites, EcoRI and HindIII can be in the reverse order to that shown.) 
     SS-cyt/C, codes for the complete precursor of the cytochrome b 5  including the tail portion of the haemoprotein. 
     Referring now to FIG. 7, the figure illustrates the scheme of appendage of alkaline phosphatase signal to rat liver ER cytochrome b 5  in plasmid constructions pAA-cyt, pSEC-cyt/C and pSEC-cyt/N. The full-length synthetic cytochrome b 5  gene, contained within the PstI-EcoRI sites and carrying an in-built ribosomal binding site, was cloned within the polylinker site of pBluescript SK+ to give pBLUE-cyt. In step 1, the unique PstI site, located upstream of the Shine/Dalgarno sequence of cytochrome b 5  gene was displaced immediately adjacent to the cytochrome b 5  methionine initiator codon by replacing PstI-AatII deletion with the 27 bp synthetic oligonucleotide duplex. This gave plasmid pA-cyt. Icyt. In step 2, insertion of the modified BamHI-HindIII-cleaved cytochrome b 5  gene (from pA-cyt) into identically-cleaved pFOG402. This yielded plasmid pAF-cyt in which cytochrome b 5  is placed under the control of phoA promoter but in an unphased reading frame with the alkaline phosphatase signal sequence. In step 3, involving construction of pAA-cyt, it shows the generation of the correct reading frame between the signal sequence and cytochrome b 5  by flush-ending PstI-BamHI- cleaved PAF-cyt and recircularising the plasmid. In this process the 21 residue signal sequence was installed in a contiguous reading frame with the mammalian cytochrome b 5  such that the +1 arginyl residue of mature alkaline phosphatase became included. This was to enhance the export properties of the cytochrome b 5  apo-protein. To minimise lethality to the host, the chimera was placed under tight transcriptional control of the native pho promoter in plasmid pAA-cyt, inducible by growth of the bacteria in a phosphate-limited medium. In step 4, the plasmid was further modified by appending the tail portion (T) of cytochrome b 5 . Finally, translocation of EcoO109-cleaved cytochrome b 5  -T segment into identically cleaved PUC19 gave plasmid PSEC-cyt/C. 
     Plasmid PSEC-cyt/N was derived by excising EcoRI-HindIII fragment from PAF-cyt containing pho-A-ss-b 5  and cloning into identically cleaved pUC19. 
     Referring now to FIG. 8, an expression vector as illustrated in FIG. 2 was modified by appending in fusion at the C-terminus of cytochrome b 5  with a gene sequence coding for the transit peptide of nitrite reductase (step 1). The modified plasmid (pCYT-nTP) was introduced into the cytoplasmic region of E.coli TB-1 (Lac) cells and the corresponding chimeric protein comprising the cytochrome b 5  apo-protein fused at its C-terminus with nitrite reductase transit peptide (pre-form apo-form of cytochrome b 5  -nitrite reductase transit peptide), was expressed in the cytoplasm (step 2). The pre-form chimeric protein was processed (apo form of cytochrome b 5  -nitrite reductase transit peptide) and after being translocated into the periplasmic region of the E.coli cells (step 3), it combined with the haem-prosthetic group to give the holo-protein (holo form of cytochrome b 5  -nitrite reductase transit peptide). 
     The crude recombinant chimeric protein was extracted from the pink bacteria (20 ml culture) by an osmotic shock technique (step 4). The technique involved suspending the cells in 1 ml of 20% sucrose, 0.3M Tris-HCl (pH 8), 1 mM Na 2  EDTA (STE) and incubation at room temperature for ten minutes. The cells were harvested as pellet by centrifugation at 5,000 g for six minutes and resuspended in residual STE volume. The plasmolysed cells were osmotically shocked by suspending in a minimum possible volume (approximately 5 ml) of ice-cold 0.5 mM MgCl 2  and left on ice for 10 minutes, followed by centrifugation at 10,000 g for ten minutes to yield a supernatant comprising the periplasmic fraction of the cells. 
     The chimera traced by its bright pink colour was purified by a single-step affinity chromatography (step 5), such as DEAE Sepharose column chromatography or haem-affinity chromatography. The chimera was then selectively cleaved (step 6) by CNBr treatment to generate authentic nitrite reductase transit peptide. 
     The 21 residue signal sequence is installed in a contiguous reading frame with the mammalian cytochrome b 5 , the +1 arginyl residue of mature alkaline phosphatase being included to enhance the export properties of the apo-protein chimera. 
     Referring now to FIG. 9, an expression vector as illustrated in FIG. 3 was modified by inserting in a contiguous reading between the signal sequence and cytochrome b 5 , a BamHI-PstI synthetic gene coding for nitrite reductase transit peptide (step 1). The derivative plasmid (pnTP-cyt) was introduced into the cytoplasmic region of E.coli TB-1 (Lac) cells and the corresponding chimeric protein comprising the pre-form apo-form of nitrite reductase transit peptide-cytochrome b 5  was expressed in the cytoplasm (step 2). The processed form of the chimeric protein (apo-form of nitrite reductase transit peptide-cytochrome b 5 ) was then translocated into the periplasmic region of the E.coli cells (step 3), where combination with the haem-prosthetic group occurred constituting the holo-protein. 
     The crude recombinant protein was extracted from the pink bacterial (20 ml culture) by an osmotic shock technique (step 4). The technique involved suspending the cells in 1 ml of 20% sucrose, 0.3M Tris-HCl (pH 8), 1 mM Na 2  EDTA (STE) and incubation at room temperature for ten minutes. The cells were harvested as pellet by centrifugation at 5,000 g for 6 minutes and resuspended in residual STE volume. The plasmolysed cells were osmotically shocked by suspending in a minimum possible volume (approximately 5 ml) of ice-cold 0.5 mM MgCl 2  and left on ice for 10 minutes, followed by centrifugation at 10,000 g for ten minutes to yield a supernatant comprising the periplasmic fraction of the cells. 
     The chimera traced by its bright pink colour was purified by a single-step affinity chromatography (step 5), such as DEAE Sepharose column chromatography or haem-affinity chromatography. The nitrite reductase transit peptide was cleaved from the isolated chimera by CNBr. 
     The 21 residue signal sequence is installed in a contiguous reading frame with the foreign gene and the subsequent mammalian cytochrome b 5 . The +1 arginyl residue of mature alkaline phosphatase being included to enhance the export and processing characteristics of the chimeric apo-protein. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- (1) GENERAL INFORMATION:-    (iii) NUMBER OF SEQUENCES: 3- (2) INFORMATION FOR SEQ ID NO: 1:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 495 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: circular-     (ii) MOLECULE TYPE: DNA (genomic)-     (vi) ORIGINAL SOURCE:#coli     (A) ORGANISM: Escherichia     (B) STRAIN: TB-1#1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GTCAGTAAAA AGTTAATCTT TTCAACAGCT GTCATAAAGT TGTCACGGCC GA - #GACTTATA  60- GTCGCTTTGT TTTTATTTTT TAATGTATTT GTACATGGAG AAAATAAAGT GA - #AACAAAGC 120- ACTATTGCAC TGGCACTCTT ACCGTTACTG TTTACCCCTG TGACAAAAGC CC - #GGATGGCT 180- GAACAAAGCG ACAAAGACGT CAAATACTAC ACTCTGGAAG AAATCCAAAA AC - #ACAAAGAC 240- TCGAAGTCGA CGTGGGTGAT CCTGCACCAT AAAGTATACG ACCTAACTAA AT - #TCCTCGAA 300- GAGCACCCCG GGGGCGAAGA AGTCCTGAGA GAACAGGCCG GCGGTGACGC GA - #CTGAAAAC 360- TTCGAAGACG TTGGCCATAG TACCGACGCT CGAGAACTGT CGAAAACGTA CA - #TCATCGGT 420- GAGCTGCACC CGGACGATCG TTCTAAAATC GCGAAACCGT CCGAAACTCT GT - #AATGAGAA 480#   495- (2) INFORMATION FOR SEQ ID NO: 2:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 3918 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: circular-     (ii) MOLECULE TYPE: DNA (genomic)#2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GA - #GACGGTCA  60- CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TC - #AGCGGGTG 120- TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CT - #GAGAGTGC 180- ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC AT - #CAGGCGCC 240- ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TC - #TTCGCTAT 300- TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA AC - #GCCAGGGT 360- TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT AC - #CCGGGGAT 420- CCTCTAGAGT CGACCTGCAG GCATGCAAGC TTGGCGTAAT CATGGTCATA GC - #TGTTTCCT 480- GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CA - #TAAAGTGT 540- AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CT - #CACTGCCC 600- GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA AC - #GCGCGGGG 660- AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GC - #TGCGCTCG 720- GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GT - #TATCCACA 780- GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GG - #CCAGGAAC 840- CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CG - #AGCATCAC 900- AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG AT - #ACCAGGCG 960- TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TA - #CCGGATAC1020- CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CT - #GTAGGTAT1080- CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CC - #CCGTTCAG1140- CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AA - #GACACGAC1200- TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TG - #TAGGCGGT1260- GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AG - #TATTTGGT1320- ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TT - #GATCCGGC1380- AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TA - #CGCGCAGA1440- AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TC - #AGTGGAAC1500- GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CA - #CCTAGATC1560- CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AA - #CTTGGTCT1620- GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT AT - #TTCGTTCA1680- TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CT - #TACCATCT1740- GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TT - #TATCAGCA1800- ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT AT - #CCGCCTCC1860- ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TA - #ATAGTTTG1920- CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TG - #GTATGGCT1980- TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GT - #TGTGCAAA2040- AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CG - #CAGTGTTA2100- TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CG - #TAAGATGC2160- TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GC - #GGCGACCG2220- AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AA - #CTTTAAAA2280- GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT AC - #CGCTGTTG2340- AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TT - #TTACTTTC2400- ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GG - #GAATAAGG2460- GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AA - #GCATTTAT2520- CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TA - #AACAAATA2580- GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC CA - #TTATTATC2640- ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTTCA AG - #AATTCTCA2700- TGTTTGACAG CTTATCATCG ATAAGCTAGC TTTGGAGATT ATCGTCACTG CA - #ATGCTTCG2760- CAATATGGCG CAAAATGACC AACAGCGGTT GATTGATCAG GTAGAGGGGG CG - #CTGTACGA2820- GGTAAAGCCC GATGCCAGCA TTCCTGACGA CGATACGGAG CTGCTGCGCG AT - #TACGTAAA2880- GAAGTTATTG AAGCATCCTC GTCAGTAAAA AGTTAATCTT TTCAACAGCT GT - #CATAAAGT2940- TGTCACGGCC GAGACTTATA GTCGCTTTGT TTTTATTTTT TAATGTATTT GT - #ACATGGAG3000- AAAATAAAGT GAAACAAAGC ACTATTGCAC TGGCACTCTT ACCGTTACTG TT - #TACCTGTA3060- AAATCCTGTG ACAAAAGCCC GGATGGCTGA ACAAAGCGAC AAAGACGTCA AA - #ATAAAGTA3120- AGGAAATACT ACACTCTGGA AGAAATCCAA AAACACAAAG ACTCGAAGTC GA - #CGTTTGGG3180- ATTGGGTGAT CCTGCACCAT AAAGTATACG ACCTAACTAA ATTCCTCGAA TA - #ATATGGAG3240- CACCCCGGGG GCGAAGAAGT CCTGAGAGAA CAGGCCGGCG GTGACGGGGG AA - #GGGAAGGA3300- GCGACTGAAA ACTTCGAAGA CGTTGGCCAT AGTACCGACG CTCGAGAACT GT - #CGAAAACG3360- TACATCATCG GTGAGCTGCA CCCGGACGAT CGTTCTAAAA TCGCGAAACC GT - #CCGAAACT3420- CTGATCACTA CCGTTGAATC GAACTCTAGT TGGTGGACTA ACTGGGTTAT CC - #CTGCGATC3480- TCTGCTCTGG TTGTAGCGCT GATGTACCGT CTGTACATGG CTGAAGATTA AT - #GAAAATTA3540- GTTAAGAGAA TTCGATATCA AGCTTTAGTT CGTCAAGGCT TGGCTAAAGT TG - #CTTATGTT3600- TACAAACCTA ACAATACATA TGAACAACAT TTAAGAAAAA GTGAAGCACA AG - #CGAAAAAA3660- GAGAAATTAA ATATTTGGAG CGAAGACAAC GCTGATTCAG GTCAATAATG CT - #CATTGTAA3720- AAGTGTCACT GCTGCTAGTG GCACTTTTAT AATTTTTAGA TCCTCTACGC CG - #GACGCATC3780- GTGGCCGGCA TCACCGGCGC CACAGGTGCG GTTGCTGGCG CCTATATCGC CG - #ACATCACC3840- GATGGGGAAG ATCGGGCTCG CCACTTCGGG CTCATGAGCG CTTGTTTCGG CG - #TGGGTATG3900#3918              TC- (2) INFORMATION FOR SEQ ID NO: 3:-      (i) SEQUENCE CHARACTERISTICS:#pairs    (A) LENGTH: 511 base     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: circular-     (ii) MOLECULE TYPE: DNA (genomic)#3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GTCAGTAAAA AGTTAATCTT TTCAACAGCT GTCATAAAGT TGTCACGGCC GA - #GACTTATA  60- GTCGCTTTGT TTTTATTTTT TAATGTATTT GTACATGGAG AAAATAAAGT GA - #AACAAAGC 120- ACTATTGCAC TGGCACTCTT ACCGTTACTG TTTACCCCTG TGACAAAAGC CC - #GGATCCCC 180- CGGGCTGCAG ATGGCTGAAC AAAGCGACAA AGACGTCAAA TACTACACTC TG - #GAAGAAAT 240- CCAAAAACAC AAAGACTCGA AGTCGACGTG GGTGATCCTG CACCATAAAG TA - #TACGACCT 300- AACTAAATTC CTCGAAGAGC ACCCCGGGGG CGAAGAAGTC CTGAGAGAAC AG - #GCCGGCGG 360- TGACGCGACT GAAAACTTCG AAGACGTTGG CCATAGTACC GACGCTCGAG AA - #CTGTCGAA 420- AACGTACATC ATCGGTGAGC TGCACCCGGA CGATCGTTCT AAAATCGCGA AA - #CCGTCCGA 480#         511      TTCG ATATCAAGCT T__________________________________________________________________________