CA1337180C - Dna sequence participating in the regulation of the expression of a dna sequence coding for a precursor of a polypeptide, expression vectors and process for the periplasmic production of the polypeptide - Google Patents

Abstract

A DNA sequence of the formula 5' TTXTTCGCG 3', in which X = A, C, G or T, participates in the regulation of the expression of a DNA sequence coding for a precursor of a polypeptide. Expression vectors contain such a sequence. The periplasmic production of a polypeptide is effected in a strain of Gram-negative bacteria transformed by one of these vectors, particularly the periplasmic production of human growth hormone.

Description

nN~ .~e~lence p~rt; c; DAt; n~ ; n the re~ul~t;on of the expre~;on of A DN~ ~eql]ence co~;no for ~ ~recl~rsor of A pol ypepti~e. expre~ion vector~ ~n~ proce~ for the peripl~;c pro~l]ction of the ~olypepti~e 05 The invention relates to a DNA ~equence partici-pating in the regulation of the expression of a DNA
sequence coding for a precursor of a polypeptide. It further relates to the expression vectors into which such a sequence is introduced. It further relates to the process which consists in expressing these vectors in a strain of Gram-negative bacteria for the periplasmic pro-duction of a polypeptide. It relates more particularly to a process for the production of human growth hormone.
It is known that Gram-negative bacteria, and more particularly strains of the species ~cher;ch;A col;, are preferred hosts for the production of heterologous poly-peptides, i.e. polypeptides which are not normally syn-thesized by the strain used, and especially polypeptides of eukaryotic origin. They are suitable for the produc-tion of polypeptides synthesized in the form of a pre-polypeptide called a precursor. In fact, only the mature form pa~ses through the cell membrane and accumulates in the periplasm, from which it can be extracted simply by an osmotic shock. The purification of the polypeptide obtained in this way is simplified becau~e it is a major component of the periplasm.
The advantage~ of such a production are rendered all the more appreciable the greater the efficiency of the transcription of the DNA sequence coding for said 3~ precursor, the translation of the messenger RNA and then the passage of the mature polypeptide through the cell membrane.
Ways of improving the transcription have been widely studied. Improvement involves des;~n;ng high-strength promoter sequences which are easy to control.

~!

- 2 - 1~37 180 European patent applications A-067540 and A-018069 relate to the protection of ~uch sequences.
A few studies have been devoted to ways of im-proving the translation Thus European patent applica-05 tion A-O 241 446 has disclosed novel DNA sequences located upstream from the Shine-Dalgarno sequence, the transcription of which yields a sequence on the messenger RNA hybridizing specifically with nucleotides 447 to 487 of ribosomal RNA 16S (or rRNA 16S), which is an essential constituent of the ribosomes. The resulting modification of the ribosome binding site is said to make the trans-lation more efficient for heterologous proteins synthe-sized in the form of an N-methionylated mature protein accumulating in the bacterial cytoplasm.
The applicant checked whether one of the pre-ferred constructions described in European patent appli-cation A-O 241 446 would be suitable for the production of polypeptides localized in the periplasm. For lack of a satisfactory result, it attempted to introduce, up-stream from the Shine-Dalgarno sequence, other DNA
sequences whose transcription would yield a messenger RNA
sequence capable of hybridizing with rRNA 16S. There proved to be a large number of such sequences, but fsw of them had the expected efficacy. On the other hand, this research led the applicant to identify a sequence of great interest.
According to a first a~pect, the invention relates precisely to a DNA sequence participating in the regulation of the expression of a DNA sequence coding for a precursor of a polypeptide, which regulatory DNA
~eguence contain~ n nucleotides, n being between 4 and 10, at least n-1 of these nucleotides being complementary to nucleotides 970 to 978 of rRNA 16S.
The ribosomal RNA 16S sequence in question ha~
formula (a):

3' AAGAAGCGC 5 in which A = Adenine monophosphate C = Cytosine monophosphate 05 G = Guanine monophosphate The regulatory sequence according to the inven-tion advantageously has formula (b):

in which T = Thymine monophosphate C = Cytosine monophosphate G = Guanine monophosphate Seguence (b) i8 complementary to sequence (a) for 8 of it~ 9 nucleotides, its transcription determines, on the corresponding messenger RNA, a sequence 5 UUUUUCGCG
3 (U = Uracil) which is complementary for 8 of its 9 nucleotides to the ribosomal RNA 16S seguence between nucleotides 970 and 978, 5' CGCGAAGAA 3. The efficacy of this regulatory sequence (b) is all the more unexpected because it is known that nucleotides 972 to 975, CGAA, of ribosomal RNA 16S are normally involved in a bond with nucleotides 960 to 963, W CG, of said RNA so as to con-tribute to the stabilization of it~ ~econdary structure (H.F. NOLLER, Science, 212 (1981) 403-411).
The invention is not limited to this particular DNA sequence TTTTTCGCG. It further relates to any sequence of the type TTXTTCGCG, in which X = A, C or G, and any ~equence which is derived from the latter and contains a series of at least 4 of its nucleotides, on condition that, in the corresponding messenger RNA
sequence, 4 of the nucleotides hybridize with nucleotides 970 to 978 of rRNA 16S.
The sequence according to the invention is loca-ted upstream from the Shine-Dalgarno sequence stricto sensu. It must not be more than 10 nucleotides away from it. Preferably, it is separated from it by only 4 nucleotides.
The other regulatory sequences located on either side of the sequence according to the invention can take 05 the known forms. In particular, the nature of the pro-moter is of little importance.
According to another aspect, the invention relates to the expression vectors into which a sequence coding for a precursor of a polypeptide, and located downstream from and under the control of a regulatory sequence containing a sequence according to the inven-tion, has been inserted.
These expression vector~ can be of any kind and are preferably plasmids.
According to another aspect, the invention relates to a process for the periplasmic production of a polypeptide. A characteristic feature of this process, which consists in transforming a strain of Gram-negative bacteria with an expression vector carrying a sequence coding for a precursor of a polypeptide, and in cultiva-ting the transformed bacteria so as to permit the expres-sion of this sequence and the maturation of the precursor by passage through the cytoplasmic membrane of the mature polypeptide accumulating in the periplasm, is the use of a vector carrying a sequence according to the invention.
The nature of the bacterial strain is of little importance. It appears in particular that strains carrying a mutation affecting at least one or other of the cya and crp genes, such as the strains described in European patent application A-0245138, are suitable.
The process according to the invention is approp-riate for the production of polypeptides synthesized in the form of a precursor. The sequence coding for the precursor can correspond either to a natural sequence or 3~ to a sequence which has been wholly or partially modified as regards its part coding for the signal peptide. It can also be a hybrid sequence in which a ~equence coding for a natural or unnatural signal peptide is associated with a sequence coding for the mature polypeptide.
Q5 The process according to the invention is par-ticularly appropriate for the production of polypeptides which are heterologous relative to the strain used, especially polypeptides of eukaryotic origin.
In one embodiment, the invention relates pre-cisely to a process for the production of human growth hormone lor hGH).
The preqent invention will now be de~cribed in greater detail with the aid ofthe following example in which reference will be made to the three Figures attached.
Figure 1 shows a restriction map of plasmid pl63~1. The different restriction ~egments are labeled arbitrarily according to the following legend:

_______ ~ ____ = Localization of the origin of replication (ORI).

_______________ = DNA segment derived from plasmid pBR322.

= DNA segment containing the sequence coding for a natural precursor of hGH.

.'. . .'.'.- = DNA segment of phage fd containing . . ~
a tran~cription terminator.
/l/////////~ = DNA segment containing a UV5 trypto-phan-lactose hybrid promoter-operator.

1~371~0 = DNA segment coding for ~-lactamase (ApR: ampicillin resistance).

Figure 2 shows the restriction map of a plasmid 05 whose PvuI-XhoI-BamHI(1) and PvuI-ORI-BamHI(2) fragments originate from plasmids pl63,1 and pBR327 respectively and whose small BamHI(2)-BamHI(1) fragment is fragment 3 described in Example 1 below.
Figure 3 shows a restriction map common to plas-mids p380,1 and p373,2. The different restriction seg-ments are labeled arbitrarily according to the following legend;

= PvuI-BamHI sequence derived from _+_+_+_ lS plasmid pBR327.
~~~~~~~ = PvuI-XhoI se~uence derived from plasmid pl63,1.
l/l/lll = XhoI-HincII sequence derived from plasmid pl63,1.
NdeI PstI
(HincII ) I I = Fragment 4 described in Example 1 below.

XXXXXXXXX = Fragment 3 described in Example 1 below.

= DNA fragment of phage fd containing a transcription terminator.

RXAMPr.R Periplasmic production of human growth hormone 1. Rb~ter; A AnA pl A~i ~.~ ll~A
One bacterial strain and four plasmid~ were u~ed.
The strain used is a strain of the species R~cher;ch; A col; which is directly related to the strain described in European patent application A-0245138, deposited in the Collection Nationale de Cultures de Microorganismes (CNCM, Paris, France) on 17 February 1986 under the reference I-529. This strain carries a cya mutation by deletion and a crp mutation by deletion.
05 The plasmids are those referred to below as p380,1, p381,1, p373,2 and p371,1.
Construction of the plasmids The strategy employed utilized fragments obtained from already existing plasmids available to the public and fragments prepared by synthesis according to the techni~ues now in common use.
Plasmid pl63,1 (Figure 1), described in European patent application A-0245138 and deposited in the CNCM
under the reference I-530 on 17 February 1986, was diges-ted with the enzymes PvuI and BamHI. The PvuI-BamHI
fragment - hereafter fragment 1 - containing the action site of the restriction enzyme XhoI, shown in Figure 1, was purified.
Likewise, plasmid pBR327, well known to those skilled in the art (q.v. SOBERON, X. et al., Gene, 9 (1980) 287-305), was digested with the enzymes PvuI and BamHI. The PvuI-BamHI fragment - hereafter fragment 2 -containing the origin of replication, was purified.
Fragment 3 was then prepared; this is a synthetic BamHI(1)-BamHI(2) fragment containing the laci gene and its promoter and having the following DNA se~uence, on which the 2 ends of the strand are identified by the numbers 1 and 2 in order to specify the orientation of the fragment in the plasmids described in Figures 2 and 3:

F ~ ~ T 3 BamHI(l) 5' GATCC GCGG M GCAT MM GTGT M A GCCTGGGGTG CCTAATGAGT
GAGCTAACTT ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG
GAAACCTGTC GTGCCAGCTG CATTAATG M TCGGCCAACG CGCGGGGAGA
GGCGGTTTGC GTATTGGGCG CCAGGGTGGT TTTTCTTTTC ACCAGTGAGA
o CGGGCAACAG CTGATTGCCC TTCACCGCCT GGCCCTGAGA GAGTTGCAGC
AAGCGGTCCA CGCTGGTTTG CCCCACCACC CGAAAATCCT GTTTGATGGT
GGTT M CGGC GGGATATAAC ATGAGCTGTC TTCGGTATCG TCGTATCCCA
CTACCGAGAT ATCCGCACCA ACGCGCAGCC CGGACTCGGT MTGGCGCGC
ATTGCGCCCA GCGCCATCTG ATCGTTGGCA ACCAGCATCG CAGTGGGAAC
GATGCCCTCA TTCAGCATTT GCATGGTTTG TTG M M CCG GACATGGCAC
TCCAGTCGCC TTCCCGTTCC GCTATCGGCT G M TTTGATT GCGAGTGAGA
TATTTATGCC AGCCAGCCAG ACGCAGACGC GCCGAGACAG M CTT M TGG.
GCCCGCT M C AGCGCGATTT GCTGGTGACC C M TGCGACC AGATGCTCCA
CGCCCAGTCG CGTACCGTCT TCATGGGAGA M ATAATACT GTTGATGGGT
GTCTGGTCAG AGACATC M G AAATAACGCC GGAACATTAG TGCAGGCAGC
TTCCACAGCA ATGGCATCCT GGTCATCCAG CGGATAGTTA ATGA-TCAGCC
CACTGACGCG TTGCGCGAGA AGATTGTGCA CCGCCGCTTT ACAGGCTTCG
ACGCCGCTTC GTTCTACCAT CGACACCACC ACGCTGGCAC CCAGTTGATC
GGCGCGAGAT TT M TCGCCG CGAC M TTTG CGACGGCGCG TGCAGGGCCA
GACTGGAGGT GGC M CGCCA ATCAGC M CG ACTGTTTGCC CGCCAGTTGT
TGTGCCACGC GGTTGGG M T GT M TTCAGC TCCGCCATCG CCGCTTCCAC
TTTTTCCCGC GTTTTCGCAG MM CGTGGCT GGCCTGGTTC ACCACGCGGG
MACGGTCTG ATAACAGACA CCGGCATACT CTGCGACATC GTATAACGTT
ACTGGTTTCA CATTCACCAC CCTGAATTGA CTCTCTTCCG GGCGCTATCA
TGCCATACCG CGAAAGGTTT TGCGCCATTC GATGGTGTCC G 3' BamHI(2) Fragment~ 1, 2 and 3 were then ligated to give the intermediate plasmid described in Figure 2.
This plasmid was subjected to partial dige~tion g with the restriction enzymes HincII and PstI. The HincII-PstI fragment, containing the origin of replica-tion and shown in Figure 3, was then ligated to fragment 4 shown below, which is a synthetic DNA fragment carrying 05 a sequence coding for the first 44 amino acids of a natural precursor of hGH and, upstream from this sequence, regulatory signals FRAGM~NT 4 ClaI

5' TCGAGCTGACTGACCTGTTGCTTATATTACATCGA
AGCTCGACTGACTGGAC M CGAATATAATGTAG~T
NdeI
TAGCGTATAATGTGTGGMTTGTGAGCGGATMCAATTTCACACAGXXXXXXXXXMGAAGGAGATATACAtT
_ _ _ ATCGCATATTACACACCTT M CACTCGCCTATTGTT M AGTGTGTCXXXXXXXXXTTCTTCCTCTATATGTA

ATG GCT ACC GGA TCC CGG ACT AGT CTG CTC CTG GCT TTT GGC CTG CTC TGC CTG
______ _ _ _ ___ _ ____ __ ___ __ _ _ _ ____ T,AC CGA TGG CCT AGG GCC TGA TCA GAC GAG GAC CGA M A CCG GAC GAC ACG GAC
lM A T G S R T S L L L A F G L L C L
-XbaI
CCC TGG CTT CAA GAG GGC AGT GCC TTC CCA ACC ATT CCC TTA T~T AGA CTT TTT
______ _ _ _ _ _ _ _ _ __ _ ___ _ _ _ _ _ _ GGG ACC G M GTT CTC CCG TCA CGG AAG GGT TGG TAA GGG AAT AGA TCT GAA M A P W L Q E G S A F P T I P L S R~ L f GAC AAC GCT ATG CTC CGC GCC CAT CGT CTG CAC CAG CTG GCC TTT GAC ACC TAC
__ _ _ __ ___ _ _ _ _ __ CTG TTG CGA TAC GAG GCG CGG GTA GCA GAC GTG GTC GAC CGG AAA CT.G TGG ATC
D N A M L R A . H R L H Q L A F L T Y

PstI
CAG GAG TTT GAA G M GCC TAT ATC CCA AAG GAA CAG AAG TAT TCA TTC CTG CA
__ _ __ _ _ _ _ _ __ _ __ GTC CTC AAA CTT CTT CGG ATA TAG GGT TTC CTT GTC TTC ATA AGT M G G S' Q E F E E A Y I P K E Q K Y S f -In this fragment, the amino acids are designated by letters according to the following code:

A = alanine M = methionine 05 C = cysteine N = asparagine D = aspartic acid P = proline E = glutamic acid Q = glutamine F = phenylalanine R = arginine G = glycine S = serine H = hi~tidine T = threonine I = isoleucine V = valine K = lysine W = tryptophan L = leucine Y = tyrosine 1~ Sequences -35 (TTGCTT) and -10 (TATAAT) of the promoter sequence, and the Shine-Dalgarno sequence well known to those skilled in the art, are underlined in that order. The symbols X positioned upstream from this sequence represent the following particular series of 9 nucleotides: 5' TTTAACTTT

AAATTGAAA 5' Plas~id p380,1 was obtained in this way.
Plasmid p380,1 (Figure 3) was used to construct 3 other plasmids by replacing its ClaI-NdeI fragment, marked on fragment 4 above, with each of the following 3 ClaI-NdeI fragments:

- 11- 13371~0 Fr~ ~ment .q ClaI

05 5' CGATAGCGTATAATGTGTGGAATTGTGAGCGGATAACA
TATCGCATATTACACACCTTMCACTCGCCTATTGT

NdeI
ATTTCACACAGTCTCCCCGCMGMGGAGATATACA
TMAGTGTGTCAGAGGGGCGTTCTTCCTCTATATGTAT 5' Fr~ ement b ClaI
5' CGATAGCGTATMTGTGTGGAATTGTGAGCGGATAACA
TATCGCATATTACACACCTTAACACTCGCCTATTGT

2Q NdeI
ATTTCACACAGTCAATGAGCAAGMGGAGATATACA
TMMGTGTGTCAGTTACTCGTTCTTCCTCTATATGTAT 5' 25 Fr~ P~ent c ClaI
5' CGATAGCGTATAATGTGTGGMTTGTGAGCGGATMCA
TATCGCATATTACACACCTTAACACTCGCCTATTGT

NdeI
ATTTCACACAGTTTTTCGCGMGAAGGAGATATACA
TAAAGTGTGTCAAAAAGCGCTTCTTCCTCTATATGTAT 5' The plasmids obtained are plasmid p381,1 with fragment a? plasmid p371,1 with fragment b and plasmid p373,2 with fragment c.
2. General m~tho~oloey 05 The experiments performed consisted in cultiva-ting the host-vector systems in question, prepared beforehand (cf. '2.1), under conditions such as to give an adequate biomass (cf. i~2.2) and such that the cells subjected to induction produce hGH (cf. -r!2.3), in col-lecting the proteins eontained in the periplasmic space by osmotic shock (cf ~i2.4) and in determining the peri-plasmic hGH (cf. ~i2.5).
2.1 Prep~r~tion of the h~t-vector ~v~tem~
The host-vector systems were prepared according to the bacterial transformation techniques known to those skilled in the art, which are described especially in the following books:
- Molecular cloning - A Laboratory Manual - T. Maniatis, E.F. Fritsch and J. Sambrook - Cold Spring Harbor Laboratory - 1982.
- Experiments in Molecular Cenetics - J.H MILLER - Cold Spring Harbor Laboratory - 1972.
2.2 Cl~ltllre a) Inocu~tion An isolated colony obtained on a solid medium (LB
medium + agar-agar) was suspended in 5 ml of a medium (LB
medium).
The LB medium used has the following characteris-tics:
- its components introduced before autoclaving are:
~actotryptone 10 g yeast extract 5 g sodium chloride 5 g distilled water qs 1 l - its pH is adjusted to 7.3 before autoclaving;

-- ampicillin is added after autoclaving at a rate of 100 g/ml.
b) Incl~bAtion The suspension prepared in a was incubated at 05 37C for 18 h in order to allow the culture to reach the stationary growth phase. The dense suspension obtained was diluted in LB medium to give an optical density value close to 0.03 when read at 600 nm - OD at 600 nm - and 25 ml of this bacterial suspension were then incubated at 37C, with agitation, until the OD at 600 nm was of the order of 0.3.
2.3 In~ ~tion Isopropyl-l~-D-thiogalactose (or IPTG) was added to the bacterial suspension obtained according to 2.2.b in an amount such that its final concentration was egual to 1 mM, IPTG was used here to initiate and maintain the synthesis of the precursor of hGH by neutralizing the action of the repressor which normally binds to the lactose operator.
The suspension, with IPTG added, was agitated at 37C for 2 h 30 min.
2.4 Osmot;c ~hnrk Reference was made to the protocol described by N.G. NOSSAL and L.A. HEPPEL in 'The Journal of ~3iological Chemistry, 241 (1966) 3055-3063 .
a) W~h;n~ w;th tr;~ ~n~ ~nTA
A sample of the suspension as obtained in 2.3 after induction was taken and centrifuged for 5 minutes at 6000 g.
The residue was taken up in a volume of buffer at pH 7 (solution A) (cf. above) such that the suspension obtained had an OD at 600 nm of the order of 10.
The buffer used was prepared by adding the fol-lowing to distilled water:
tri(hydroxymethyl)aminomethane-HCl, or tris-HCl, added so as to give a final concentration of 30 mM.
ethylenediaminetetraacetic acid, or EDTA, added so as to give a final concentration of 1 mM.
b) Act;on of ~l]cro.~e 05 The suspension obtained in 2.4.a was centrifuged for 5 minutes at 6000 g.
The residue was taken up very carefully, at con-stant volume, in a solution B prepared for immediate use and corresponding to solution A to which sucrose has been added at a rate of 15 g per 100 ml.
The suspension was left for 10 minutes at 20C.
It was then centrifuged for 5 minutes at 6000 g.
The centrifuge tubes were placed in melting ice.
The supernatant was carefully removed and re-placed (at constant volume) with deionized water which had been cooled beforehand to the temperature of melting ce .
The suspension prepared in this way (having an OD
at 600 nm of the order of 10) was left for 5 minutes at 0C.
c) Collect;on of the protein.~ 1OCA1 ;7e~ in the peripl~m The suspension obtained in 2.4.b was centrifuged for lO minutes at 18,000 g.
The supernatant, which contained the proteins localized in the peripla~m, was collected.
2.5 netermi n~ti f~n of ~h~ Deri pl F~mi c hl'~
The supernatant obtained in 2.4.c was subjected to high pressure liquid chromatography using an apparatus equipped with a calibrated injection system and a detec-tor set at 220 nm.
The following were used:
a C8 - 300 Angstrom reversed-phase column made of steel, with a length of 10 cm and an internal diameter of 4.6 mm (SYNCHROM reference C-8 R103-10), a mobile phase consisting of a linear gradient passing from 70 volumes of solution S1 and 30 volumes of solution S2 to 40 volumes of solution S1 and 60 volumes of solution S2 in 20 minutes.
05 Solutions S1 and S2 had the following charac-teristics:
S1 = purified water containing 0.1% (v/v) of trifluoro-acetic acid, S2 = acetonitrile for HPLC, containing 0.08% (v/v) of trifluoroacetic acid, the flow rate was 1 ml per minute.
The optical density of the fractions was measured and the amount of periplasmic hGH, expressed in micro-grams per ml of supernatant, was determined by comparison with a previously established standard scale.
2.6 AnAly.~;~ by ~hR W~ter~ Rlot te~hn;al~
The following operations were carried out in succession:
- separation by gel electrophoresis (according to the protocol described by LAEMMLI, U.K., Nature, 22~ (1970) 680-685) of the different protein~ contained in each of the supernatants obtained according to 2.4.c; the gel used was a polyacrylamide gel (15% w/v) containing 0.5% of ~odium dodecylsulfate.
- transfer of said proteins contained in the gel on to a nitrocellulose filter (according to the technique of H. TOWBIN et al., Proc. Natl. Acad. Sci. USA, ~ (1979) 4350-4354)-- immunodetection performed according to the technique of BURNETTE (W W BURNETTE, Anal Biochem., 11~ (1981) 195-203); thi~ entails the following successive operations:
rinsing the nitrocellulose filter for 10 min with a buffer A (tris-HCl 10 mM, NaCl 170 mM, KI 1 mM), bringing the nitrocellulose filter into contact with a buffer B (buffer A with bovine ~erum albumin added at a rate of 3 g per 100 ml) for 30 min at 37C, bringing the nitrocellulose filter into contact with an immune serum (a polyclonal antibody recognizing 05 mature hGH and its precursor) for 18 h at 20C, rinsing the nitrocellulose filter with buffer B, bringing the nitrocellulo~e filter into contact with a solution of protein A labeled with iodine 125 at a rate of 0.1 microcurie per ml, for 6 h at 20C, rinsine the filter with buffer A, drying the filter between two ab~orbent sheets, bringing the filter into contact with an X-ray film, developing the film.
3. Re - Presentation It was noted that, under the chosen operating conditions, periplasmic hGH was present in its mature form for about 98% of the molecules, irrespective of the bacterial strain and the plasmid u~ed.
The re~ult~ of the determination are reported in the Table below:

Plasmid tested 371,1 373,2 380,1 381,1 1 OD at 600 nm, measured on the sus-pension obtained in 1.38 1.32 0.30 1.38 2.3 after induction 2 Peripla~mic hGH
mea~ured in micro-gram~ per ml of supernatant collected 0.73 2.13 3.56 0.24 after o~motic shock and brought to a turbidity such that OD at 600 nm = 1 - Interpretation a) Row 1 reflects the growth rate of the bacterial cells.
It is clearly apparent that plasmid 380,1 prohibits appropriate cell growth.
05 b) Row 2 reflects the capacity of each cell to produce periplasmic hGH. The result obtained with plasmid 380,1 cannot be taken into account in view of the con-clu~ion noted above. It is clear that, of the other 3 plasmids, it is plasmid 373,2 which offers the best performance characteristics by affording a production almost 10 times and 3 times greater than that afforded by plasmids p381,1 and p371,1 respectively.

Claims (8)

1. A DNA sequence participating in the regulation of the expression of a DNA sequence coding for a precursor of a polypeptide, said DNA sequence being located upstream from a Shine-Dalgarno sequence and having the formula:
5' TTXTTCGCG 3' in which X = A, C, G or T
in which:
A = Adenosine monophosphate C = Cytidine monophosphate G = Guanosine monophosphate T = Thymidine monophosphate

2. An expression vector carrying a first DNA sequence coding for a precursor of a heterologous polypeptide, and further comprising a second DNA sequence participating in the regulation of said DNA sequence coding for a precursor of a heterologous polypeptide, said second DNA sequence being located upstream from a Shine-Dalgarno sequence and having the formula:
5' TTXTTCGCG 3' in which X = A, C, G or T
in which:
A = Adenosine monophosphate C = Cytidine monophosphate G = Guanosine monophosphate T = Thymidine monophosphate

3. The vector as claimed in claim 2, wherein said second DNA sequence not being more than 10 nucleotides away from the AGGA Shine-Dalgarno sequence.

4. The vector as claimed in claim 2 or 3, which is a plasmid.

5. A process for the periplasmic production of a polypeptide, which comprises transforming a strain of Gram-negative bacteria with an expression vector carrying a sequence coding for a precursor of a polypeptide as claimed in claim 2 or 3, and cultivating the transformed bacteria so as to permit the expression of the sequence coding for the precursor and the maturation of the precursor by passage through the cytoplasmic membrane with the mature polypeptide accumulating in the periplasm.

6. The process for the periplasmic production of human growth hormone by the process claimed in claim 5.

7. The process claimed in claim 5, in which the strain of Gram-negative bacteria is an E. coli strain.

8. The process claimed in claim 7, in which the E. coli strain carries a cya mutation by deletion and a crp mutation by deletion.