Gene-dose-dependent expression of soluble ... - Springer Link

Appl Microbiol Biotechnol (1992) 38:77-83

Applied Microbiology Bioteehnology © Springer-Verlag 1992

Gene-dose-dependent expression of soluble mammalian cytochrome bs in Escherichia coli Joseph Gallagher, Naheed Kaderbhai, and Mustak A. Kaderbhai Department of Biochemistry,School of Life Sciences, The UniversityCollege of Wales, Penglais, AberystwythSY23 3DD, UK Received 27 March 1992/Accepted 23 June 1992

Summary. A synthetic structural gene encoding a mammalian cytochrome bs, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n = 1) to six (n = 6) gene copies. The gene, placed in p2-ncyt under the control of the 2PL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome bs. The expressed levels of cytochrome b5 in Escherichia coli p2ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing p2-6cyt produced 75 ~tg cytochrome b~/ml of unit optical density at 600 nm culture, constituting 55°7o of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the holocytochrome bs. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b5 in E. coli.

Introduction Cytochrome b5 (cyt) is a small haemoprotein of end~plasmic reticular (ER) origin. It acts as an electron donor in a number of reactions including fatty acid desaturation in animal liver, methaemoglobin reduction in erythrocytes and cytochrome P-450 reduction (Schenkman et al. 1976; White and Coon 1980). The liver cyt is com-

Correspondence to: M. A. Kaderbhai

posed of 133 amino acid residues arranged in two domains, an N-terminal (99 residues), catalytically active, haem-associated globular 'head' and a smaller C-terminal, hydrophobic domain that anchors the protein in the ER membrane facing the cytosol (Spatz and Strittmatter 1971). Using the soluble core of cyt as a model protein, we have focused on understanding features of recombinant protein production in Escherichia coli from an expression vector carrying tandemly polymerised genes. Firstly, can gene amplification enhance the production of a recombinant protein (Wanatabe et al. 1986; Tsung et al. 1989; Shibui et al. 1988, 1989)? The proposition that an increase in transcript units through gene amplification should elevate the intracellular level of accumulatable protein is attractive in theory. However, this may not be the case if consequent variations in the rates of translation and protein turn-over ensue. Secondly, can tandem repeats of gene units be stably propagated without their loss through recombination events (Balbas et al. 1988)? Thirdly, what is the limit of the productivity of a foreign protein in E. coli cultivated under standard batch growth conditions? Fourthly, as has been reported for a number of recombinant proteins (Schein 1989), would hyperexpression employing a high-copy-number plasmid, transcribed under a powerful promoter and at higher temperatures (above 37 ° C), result in the formation of insoluble, non-functional cyt? Lastly, in the case of over-expression of soluble cyt, can E. coli cope with the supply of de-novo-synthesised haem prosthetic group to generate the haem-assembled cyt? We have, therefore, investigated the capacity of E. coli to express the mammalian cyt protein from constructed plasmids carrying tandemly amplified structural cyt gene ranging from one to six copies, placed under the control of the )~ promoter. We report on the characteristics of the production and the properties of the recombinant cyt formed under the standard batch growth conditions at an inducible temperature at 38.5 ° C. We show that the productivity of recombinant cyt is clearly a function of gene dose. Neither hyperexpression, driven through one of the most powerful promoters at an ele-

78 vated temperature, nor batch-supply o f nutrition appear to limit the productivity o f the soluble cyt. The only exception was that with increasing cyt gene dose, the bacterial capacity to cope with haem supply becomes limiting and so largely results in the p r o d u c t i o n o f a p o p r o rein, which, however, is converted into the functional holo f o r m by exogenous supplementation with the prosthetic group.

Materials and methods

Materials. LabM (Bury, UK) supplied Tryptone and yeast extract. All other chemicals and reagents were from Sigma (Poole, UK) and BDH (Poole, UK). Restriction endonucleases and molecular biologicals were from Anglian Biotechnology (Colchester, UK).

Bacterial strains, plasmids and DNA manipulation. The host strains used throughout were E. coli TB-1, [F ara A (lac-proAB) rps q~80d laeZAM 15hsd R17 (r2 m~-)], RR-1 IF- hsdS20 (rff raft) ara-14proiA leuB6 lacY1 galK2 rpsL20 (str r) xyL5 mtl-1 supE441- recA +] and N4830-1 [ f - suo his-ilv-galK- (2ehlDpgl) (2 BamN + ci857 H1]. The plasmid pVS-45 encoding the synthetic gene of the soluble core of rat liver ER cyt was a gift from Dr. S. G. Sligar, University of Illinois, USA. Plasmid pEPFxWTP used for cloning single and multiple copies of cyt was from the University of Wales Plasmid Library. Procedures involving DNA purification, digestion, agarose electrophoresis and bacterial transformation were as described previously (Maniatis et al. 1989).

Expression and analys& of cyt. E. coli N4830-1 cells harbouring plasmids p2-ncyt were grown in LB medium (0.5% yeast extract, 1% Tryptone and 1% NaC1) in the presence of 75 gg/ml of ampicillin at 30° C. At an optical density (OD) at 600 nm of 0.8, the incubation temperature was raised to 38.5°C to switch on the A promoter. At time periods, stated elsewhere, 1 ml culture aliquots were centrifuged at 5000 g for 5 rain to obtain cell pellets. For sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE), the cells, pre-suspended in 100 gl 20% (v/v)-glycerol, were mixed with an equal volume of 5% SDS, 50 mM TRIS-HC1 (pH 7.5), 67 mM dithiothreitol and 0.03% bromophenol blue and heated over a boiling water bath for 5 rain. A volume (approximately 50 gl) containing equivalent cell numbers (6.42 x 108), was taken for SDS-PAGE analysis. To obtain soluble proteins, the cells were lysed by incubating in 250 gl lysis buffer [10 mM TRISHCI, 1 mM NazEDTA (TE) containing 100 gg/ml lysozyme] for 20 min on ice. After two rapid freeze-thaw cycles and centrifugation at 15000 g for 10 min, protein from 25 ~tl of the resulting supernatant was mixed with an equal volume of loading buffer (50% (v/v) glycerol, 25 mM TRIS-HC1 (pH 7.0) and 0.03% bromophenol blue) prior to separation on a non-denaturing gel. Proteins were resolved in a polyacrylamide gel gradient (18 to 10°70) without or with 0.1% SDS (Gallagher and Smith 1991). Coomassieblue-stained protein bands were anlysed using a Quick Scan densitometer (Helena Laboratories).

Quantification of cyt. Cyt (oxidised), in bacterial lysates, was quantified from absolute absorption spectra monitored against a blank lysate prepared from a comparably grown non-recombinant E. coli N4830-1 pEPFxWTP (pX-0cyt). The spectra were monitored (at 25° C) by scanning the sample from 350 nm to 450 nm across a light path of 10 mm in 50 mM TRIS-acetate buffer, pH 8.0, and 1 mM Na2EDTA. The quantity of cyt was calculated from the Soret absorption peak at 413 nm using the absorption coefficient of 115 mM - l ' c m -1 (Hultquist 1978). Where haem was supplemented to reconstitute apocyt, it was added as a stock 1 mM solution [0.1 M TRIS-HC1 (pH 8.2), 80% (v/v) ethylene glycol] in

equal amounts to the recombinant and non-recombinant samples at final concentrations ranging from .10 to 40 gM.

Protein. Protein was assayed by the Bradford (1976) procedure using bovine serum albumin as the standard.

Results

Construction o f plasmids (p2-ncyO encoding single and multiple copies o f cyt Figure 1A shows the basic scheme o f plasmid construction. Plasmid pVS-45, a derivative o f pUC13 (Beck von B o d m a n et al. 1986), carried the complete synthetic gene encoding the soluble core o f cyt with an in-built, optimised S h i n e / D a l g a r n o (S/D) sequence, contained within the restriction sites PstI-EcoRI o f 326-bp D N A . To facilitate cyt gene duplication, a unique NsiI restriction site was fostered downstream o f the eyt gene by inserting the EeoRI-NdeI D N A fragment o f a precusor o f the small subunit o f wheat (ps) (Triticum aestivium) ribulose-l,5-bisphosphate carboxylase (Kaderbhai et al. 1990) to give the vector p L - l c y t . The PstI-NdeI cleaved cyt gene was isolated f r o m p L - l c y t and ligated into the NsiI-NdeI-restricted (larger fragment) o f p L - l c y t to yield the vector pL-2cyt coding for two t a n d e m copies o f the cyt gene. Similarly, the creation o f pL-3cyt involved relocating the smaller PstI-NdeI D N A , (containing two cyt gene copies f r o m pL-2cyt), into NsiI-NdeI-restricted p L - l c y t . Using the same strategy, we constructed pLncyt vectors containing four, five and six copies o f the cyt gene, each set under the control o f a single Lac promoter. Subseqeunt translocation o f PstI-NdeI-cleaved cyt gene copies f r o m p L - n c y t into NsiI-NdeI-cleaved p20cyt ( p E P F x W T P ) resulted in p,~-ncyt vectors h a r b o u r ing cyt gene copies under the control o f the 2PL p r o m o t er (see Fig. 1B). Plasmids p2-ncyt were p r o p a g a t e d in E. coli, initially in host RR-1 (Rec A +) and subsequently in host N48301, for over several h u n d r e d generations. Restriction m a p p i n g o f the p2-ncyt plasmids generated a profile o f cleaved fragments o f single and polymeric cyt gene copies, the sizes o f which were in agreement with the engineered scheme. This verified that no rearrangement o f the repeated sequences had occurred (see Fig. 2).

Expression o f cyt is proportional to gene dose E. coli TB-1 pL-ncyt, expressing cyt under the control o f the Lac p r o m o t e r , treated with 2 mM isopropyl-~-D-galactopyranoside, did not show any appreciable induction o f a protein comparable in size to cyt following electrophoretic analysis o f the bacterial proteins (data not shown). We next translocated the single and tandemly polymerised cyt structural gene into an 2 regulated expression vector (p)L-ncyt). T h e r m o - i n d u c t i o n o f E. coli N4830-1 cell lines carrying either one or m o r e copies o f the cyt gene resulted in bright pink bacterial cultures. Analysis o f the total bacterial proteins by S D S - P A G E

79

~I

A

lac

/~tl

~

N~I

/~

3340

_

~

bps

~I ~

Pstl ~

leyt

2cyt

Pstl

icyt

3cyt 2cyt

Pstl

lcyt

4cyt 3cyt

2eyt

PstI

lcyt

PstI

5cyt 4cyt

3cyt ~eyt

I leyt

6cyt

4eyt

2¢yt

~

~

N

~

i

l

Ndel I

~-~,,

5cyt

3cyt

lac / ~ t l

Nd¢I NdeI

Ndel NdeI

I

'

1

NMI P*tl

NdeI

I

HindllI

l

a

m

b

d

~

Fig. 1. A Scheme illustrating amplification of the cytochrome be (cyt) gene in pL-ncyt: step 1, PstI-NdeI cleavage and isolation of cyt-precursor (ps) DNA; 2, NsiI-NdeI-cut larger vector fragment carrying the cyt gene; 3, ligation of the fragments to generate pL-

2cyt. B plasmids p2-n-cyt encoding one to six cyt gene copies under control of the thermoinducible 2PL. The vectors were constructed by translocating PstI-NdeI-cut cyt gene(s) from pL-ncyt into NsiI-NdeI-cut pEPFXW (p,~-0cyt), a derivative of pBR322

80 O.B ~

0.7

~ 0.6 C; O~ 0.5

.~

~ ~.4 ~o N ~ 0.3

Ahinduced

~:~ 0.2 -~ ~ 0.~ 0 1

2

3

4

5

cyt gene copies

Fig. 4. Cyt production in bacterial cells harbouring a varying number of cyt genes. Cyt in bacterial cells was monitored spectroscopically as described in Materials and methods

protein at the cyt gene dose of one, two, three, four, five and six respectively, as analysed by densitometric scans of the resolved polypeptides. Fig. 2. Mapping of p)~-ncyt. The plasmids were doubly digested with HindIII and NdeI to dissect the cyt gene copies. The observed (and expected) sizes in bp of 1 cyt, 2 cyt, 3 cyt, 4 cyt, 5 eyt to 6 cyt fragments were 1500 (1543), 2000 (2030), 2500 (2517), 3000 (3004), 3500 (3491), and 4000 (3978), and that of the parental vector fragment was 2600 (2636)

Fig. 3. Gene-dose-dependent expression of cyt. Lysates from equivalent number of bacterial cells, thermo-induced for 4 h, were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins detected by staining with Coomassie blue R-250 staining

revealed prominent synthesis of an induced polypeptide of 12.5 kDa (Fig. 3), later identified as cyt (see below). The intensity of the cyt band, detected by Coomassie blue staining, clearly correlated with the number of cyt genes. The induced 12.5-kDa cyt band constituted 8070, 18%o, 23%o, 28%o, 31% and 34% of the total bacterial

Soluble cyt is expressed predominantly in an apoform

A spectroscopic measure of the cyt content in E. coli p2ncyt, thermo-induced for 4 h, showed that when the cyt gene dose increased from one to two the level of the recombinant protein elevated from 4.5% to 5.5% of total bacterial protein (Fig. 4). Surprisingly, with further increases in the gene dose, ranging from three to six copies, the cyt content gradually declined, eventually reaching a level of about only 2%. A small but significant improvement in the amount of the expressed cyt occurred with extension of the induction period to 19 h. Two factors could account for the significantly poorer productivity of the spectroscopically measured haemoprotein over that detected by SDS-PAGE analysis. Firstly, overproduction, through increased gene dose together with use of the run-away )~ replicon, may have led to deposition of most of the recombinant cyt in an insoluble, aggregated form (Schein and Noteborn 1988). The second possibility was that the cyt accumulated as a soluble apoprotein but lacked the haem moiety. To differentiate between these possibilities, we analysed the proteins in the soluble fraction of the bacteria expressing one to six cyt gene copies in a non-denaturing gel (Fig. 5). A major, thermo-inducible acidic protein band, identifiable as cyt by its bright brown appearance, prior to staining with Coomassie blue, was distinctly visible. The amount of this protein elevated with increasing gene dose and appeared to correspond with the gene copy number indicating that most, if not all, of the recombinant cyt was accountable in the soluble form. The brown-coloured, major induced protein from the soluble fraction of E. coli p2-3cyt, resolved on a nondenaturing gel, isolated by electroelution, was extensively dialysed against TE. The recombinant cyt had an oxidised protein Soret band at 413 nm, a reduced protein Soret increased peak at 423 nm and visible peaks at

81

E = 5 4~ O = 4

3~

~ 3 2c,~

Fig. 5. Production of soluble cyt as a function of gene dose. Soluble extracts were prepared from Escherichia coli carrying one to six copies of the cyt gene and thermoinduced for 4 h. Extract prepared from the equivalent number of bacterial cells were analysed in a non-denaturing polyacrylamide gel

1.5

,~ E = 2

lcyt

~

k~

0

45

i

~

i

i

10

20

30

40

Haem (,uM)

1.0

Fig. 7. Conversion of apocyt to holocyt by exogenous haem. Soluble lysates prepared from thermo-induded E. coli p2-ncyt were subjected to spectral analysis against a lysate (blank) prepared from a comparably grown non-recombinant E. coil harbouring pEPFxWTP (p2-0cyt). Haem, at the stated concentrations, was added to both the recombinants and non-recombinant lysates. The amount of holocyt was determined from the Soret absorbance rises at ,$13 nm

13.512.3"

abe

~ 0.5

I

350

400

I

I

I

450 500 wavelength (nm)

550

600

Fig. 6. Spectral characteristics of the purified recombinant cyt: 1, oxidised spectrum; 2, dithionite-reduced spectrum; 3, oxidised versus reduced spectrum. Inset: SDS-Page of marker proteins (a); the purified cyt used for spectral determinations (b); cytoplasmic extract of thermo-induced E. coli p2-3cyt from which the cyt was isolated (c)

555 n m and 527 nm. The absorption spectrum of the oxidised versus reduced forms of the hyperexpressed recombinant cyt was identical to that of the trypsin-digested cyt purified f r o m m a m m a l i a n tissue, in having a characteristic m a x i m u m at 423 nm and a minimum at 409 nm (Fig. 6).

H a e m supply is limiting during hyper-production o f cyt To counteract the potential deficiency of haem during the apocyt expression, the recombinant cultures were supplemented with bovine haem (5 ~tM). This treatment resulted in a 15% to 20% rise in the productivity of the

holocyt but essentially displayed a similar graphical trend to that observed in the corresponding non-haemtreated recombinant cultures, shown in Fig. 4. However, direct addition of haem to the lysed recombinant cells resulted in immediate conversion of the apocyt into its holoform, as manifested by several-fold rises (up to sixfold) in the Soret absorbance peak 413 nm. The in vitro formation of cyt was dependent upon haem concentration, reaching saturation at around 20 ~tM haem (see Fig. 7). The findings therefore establish that the bacterial capacity, to supply haem for the overproduced apocyt, irrespective of the gene dose applied, was limited to 0.5 to 1 nmol h a e m / u n i t of culture OD at 600 nm. The amount of reconstituted cyt derived is also apparently gene-dose-related, such that with an increase in gene dose from one to five copies there is a progressive increase in cyt ranging f r o m about 2 to 7 n m o l / u n i t culture OD at 600 nm. At five and six copies of the cyt gene, the productivity of the haem-reconstituted cyt was about 50°70 of the soluble bacterial protein, representing 74 ~g cyt/ml culture at an OD of 600 nm. Therefore, it can be determined that upon cyt gene duplication the amount of the in vivo haem-assembled cyt (See Fig. 4), expressed as a percentage of total combined sum of apo and holo (see Fig. 7), elevated f r o m 22°/o to 29°7o. However, at the higher gene doses (two to six) the productiv-

82 3OO [ E~2cyt ~5cyt

[

250

"~ 2o0

,~ 150

,~ ~ ~oo 50

0 ,~..0.1 ALl.0 AL5,0

GS'~ GS2 GS5

Glul

Glu2 G[U5

GS Glu 1

(raM)

Fig. 8. Effect of supplementingE. coli p~.-ncyt cultures with 5aminolevulinicacid (AL), glycine(G)/succinate (S) and glutamate (Glu) on the productivityof holocyt. The 100% absolute value is that of an untreated culture

ity of the holoprotein gradually decreased, reaching a value of only 10% at six copies of the cyt gene.

Haem synthesis in E. coil We investigated the possibility that 5-aminolevulinic acid could alleviate the haem deficiency during hyperproduction of apocyt because this compound is the first committed precursor in tetrapyrrole biosynthesis in nature (Beale 1984). Inclusion of 5-aminolevulinic acid in the culture medium caused a marked, two- to threefold stimulation in the productivity of holocyt (Fig. 8). There are two routes for the derivation of 5-aminolevulinic acid, the 'C-5 (glutamate) pathway', which is now though to be more common in the biosphere, and a route involving the condensation of succinyl CoA and glycine (Castelfranco and Beale 1983). Since the extent of the contribution of these two pathways to the synthesis of 5-aminolevulinic acid in E. coli has not yet been established, we investigated the effect of succinate/glycine and glutamate on the productivity of holocyt in the recombinant bacteria (see Fig. 8). Interestingly, both succinate (1-5 mM)/glycine (1-5 raM) and glutamate (12 5 raM) produced a significant improvement in the holocyt yield at the lower cyt gene dose. At the higher cyt gene dose, whereas succinate/glycine appears to be marginally stimulatory, glutamate inhibits the formation of holocyt.

Discussion

We have described a novel procedure for tandem polymerisation of the mammalian cyt gene. The scheme exploits repetitive use of three restriction endonuclease sites, PstI, NdeI and NsiI, that regenerate in successive plasmid constructions. Any number of cyt gene copies

(x), derived by PstI-NdeI cleavage of pL-xcyt, can be relocated into the larger vector fragment of NsiI-Ndelcleaved pL-ycyt (carrying y number of cyt gene copies). This would give rise to pL-(x+y) cyt. The strategy relies on the linkage of the compatible 5'-cohesive ends between the NsiI (parental vector) and the PstI (incoming cyt gene) sites and their concomitant apt destructions. Additional PstI (at the start of the first copy of the cyt gene) and NsiI (following the terminal cyt gene) sites reinstate in the subsequently derived vector. Each copy of the cyt gene carries its own S/D sequence embedded in an optimised ribosomal binding site. Cyt gene copies, ranging from one to six, placed under the control of a single promoter would transcribe the respective monoto homohexacistronic cyt mRNAs. The inbuilt ribosomal site within each gene would direct independent translation of the cyt protein from each cistronic unit. The tandemly polymerised cyt genes propagate stably in E. coli without any apparent rearrangement of the repeated sequences (see Fig. 2). We have demonstrated that the expression of cyt is clearly a function of the gene dose. The fact that the productivity of cyt correlates linearly with the applied gene dose indicates that the rate of transcription is the crucial determinant and the driving force in the level of recombinant protein synthesised/accumulatable under conditions where product degradation rate must be maintained at a relatively constant level (Balbas and Bolivar 1990). Hyperproduction of mammalian cyt is driven at a significantly higher gene dose than that previously reported for the expression of the prokaryotic chloramphenicol aminotransferase (Shibui et al. 1988). In this study the use of a high-level, high-copy-number expression vector together with a powerful promot-. er driven at an elevated temperature of 38.5°C still results in production of the soluble recombinant cyt. These findings are in marked contrast to the pevious reports where the use of a runaway-type 2 replicon vector at elevated temperatures (from 37 to 42 ° C) was unsuitable for production of foreign proteins due to the generation of insoluble aggregates and the induction of heatshock-associated proteases (Schein and Noteborn 1988). A likely explanation for the successful hyperproduction of cyt under these exceptional conditions may be the use of the synthetic mammalian cyt gene built on a choice of codons optimised for usage in E. coll. The case of the hyperproduced cyt may be unique in view of its exceptionally high solubility and amphiphilicity. Curiously, using the Lac promoter at a temperature of 27 to 30° C, we noticed that cyt expression was relatively poor and was not potentiated at all by gene amplification (data not presented). Under the present batch growth conditions, neither nutrition nor message stability appears to limit the productivity of cyt, although the production of the recombinant protein appears to reach a saturation point at around six copies of cyt gene. Only a proportion of the expressed cyt converts to the holoform that is spectrally identical (Fig. 6) to its native counterpart in the microsomal membranes of rat liver (Strittmatter et al. (1974); Estabrook and Werringloer (1978)). However, exogeneously-added haem con-

83 verts the remaining cell-free soluble apocyt into functional haemoprotein. The inability of exogenously supplied haem in the culture medium to restore the expected levels of holocyt suggests that the cofactor is most likely not efficiently translocated into the bacterial cells. The alleviation of haem deficiency by 5-aminolevulinic acid, added to the culture medium (see Fig. 8), indicates that haem, during the production of cytochrome b5 in E. coli, derives metabolically via the conventional de novo pathway (Jordan 1990). Therefore, the rate-limiting step(s) in haem synthesis during overproduction of cyt must accrue either at or prior to 5-aminolevulinic synthesis step. The enhanced production of the haem-assembled cyt by succinate/glycine, and to a limited extent by glutamate suggests that both the 'C-5 pathway' and the 5-aminolevulinic acid synthetase step must be operative in E. coli for the derivation of 5-aminolevulinic acid. The observation that the extent of the assembled holocyt, but not the expressed apocyt, declines with increasing copies of the cyt gene indicates that there is an apparent competition for the amino acid pool between synthesis of haem and the recombinant apocyt. The significantly faster rate of recombinant apoprotein synthesis would deplete the amino acids demanded for haem biosynthesis. This theory also explains the inability of glutamate to enhance holocyt synthesis at the higher cyt gene dose; the supplemented glutamate may fail to gain access to the 'C-5' pathway because of its rapid utilisation for the synthesis of cyt molecules. There are in fact twice as many glutamate residues (12) as glycine (6) in the cyt molecule. The ability to achieve the desired production of recombinant cyt by regulating cyt gene copies will prove to be a useful model for elucidating the pathway(s) and events that regulate haem synthesis in E. coll. We also suggest that the gene duplication approach may be useful for overcoming inadequate expression of foreign proteins in E. c o i l Acknowledgement. This work, in part, was accomplished with Support from The Agricultural and Food Research Council (PG2/ 515). We thank Dr. E. I. Mercer for Critical appraisal of the manuscript.

References BaIbas P, Bolivar F (1990) Design and construction of expression plasmid vectors in Escherichia coli. Methods Enzymol 185 : 1437 Balbas P, Soberon X, Bolivar F, Rodriguez RL (1988) The plasmid pBR322. In: Rodriguez RL, Denhardt DT (eds) Vectors. A survey of molecular cloning vectors and their uses. Butterworth, London, pp 5-41 Beale SI (1984) Biosynthesis of photosynthetic pigments. In: Bak-

er NR, Barber J (eds) Chloroplast biogenesis. Elsevier, Amsterdam, pp 133-205 Beck von Bodman S, Schuler MA, Joliie DR, Sligar SG (1986) Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome bs. Proc Natl Acad Sci USA 83 : 9443-9447 Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 Castelfranco PA, Beale SI (1983) Chlorophyll biosynthesis: recent advances and areas of current interest. Annu Rev Plant Physiol 55 : 485-490 Estabrook RW, Werringloer J (1978) The measurement of difference spectra: application to the cytochromes of microsomes. Methods Enzymol 52:212-220 Gallagher SR, Smith JA (1991) Analysis of proteins. In: Ausubel FM, Brent R, Kington R, Moore RE, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology, vol 2. Greene Publishing Associates, New York, pp 10.0.1-10.2.21 Hultquist DE (1978) Methemoglobin reduction system of erythrocytes. Methods Enzymol 52:463-473 Jordan PM (1990) The biosynthesis of 5-aminolevulinic acid and its transformation to coproporphyrinogen in animals and bacteria. In: Dailey HA (ed) Biosynthesis of heine and chlorophylls. McGraw-Hill, New York, pp 55-122 Kaderbhai MA, He M, Beechey RB, Kaderbhai NN (1990) Coexpression of a precursor and the mature protein of wheat riboluse-l,5-biphosphate carboxylase from a single gene in Escherichia coli. DNA Cell Biol 9:11-25 Maniafis T, Fritsch EF, Sambrook J (1989) Molecular cloning: a laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Schein CH (1989) Production of soluble recombinant proteins in bacteria. Bio/Technology 7 : 1141-1147 Schein CH, Noteborn MHM (1988) Formation of soluble recombinant protein in Escherichia coli is favored by lower growth temperature. Bio/Technology 6: 291-294 Schenkman JB, Jansson I, Robie-Suh KM (1976) Many roles of cytochrome b5 in hepatic microsomes. Life Sci 19:611-624 Shibui T, Uchida-Kamizono M, Teranishi Y (1988) A method for overproduction of a target gene in Escherichia coli. Agric Biol Chem 52: 2235-2241 Shibui T, Uchida-Kamizono M, Takizawa Y, Kondo J, Hiyoshi C, Marayama S, Morimoto Y, Teranishi Y (1989) High-level expression of human proapolipoprotein A-1 in Escherichia coli using expression plasmids containing tandemly polymerized prolipoprotein A-1 structural genes. Appl Microbiol Biotechnol 31:518-523 Spatz L, Strittmatter P (1971) A form of cytochrome b~ that contains an additional hydrophobic sequence of 40 amino acid residues. Proc Natl Acad Sci USA 68 : 1042-1946 Strittmatter P, Spatz L, Corcoran D, Rogers M J, Setlow B, Redline R (1974) Purification and properties of rat liver microsoreal stearoyl coenzyme A destaurase. Proc Natl Acad Sci USA 71:4565-4569 Tsung K, Inouye S, Inouye M (1989) Factors affecting the efficiency of protein synthesis in Escherichia coli. Production of a polypeptide of more than 6000 amino acid residues. J Biol Chem 264: 4428-4433 Wanatabe K, Yamano Y, Murata K, Kimura A (1986) Glutathione production with hybrid plasmids containing tandemly polymerized genes for glutathione synthetase. Appl Microbiol Biotechnol 24: 375-382 White RE, Coon MJ (1980) Oxygen activation by cytochrome P450 Annu Rev Biochem 49 : 315-356