IE861031L

IE861031L - Modified trp operon

Info

Publication number: IE861031L
Application number: IE861031A
Authority: IE
Original assignee: Hoechst Ag
Priority date: 1985-04-19
Filing date: 1986-04-18
Publication date: 1986-10-19
Also published as: IL78529A0; HU196458B; ATE44046T1; AU5638786A; HUT41068A; ZA862925B; FI861624A; DE3663956D1; JPH0665317B2; NO175646B; FI84362B; IL78529A; NO861551L; DE3514113A1; PH26596A; ES8706818A1; ES554097A0; IE58994B1; CA1321963C; DK179686D0

Abstract

For the contracting states : BE, CH, DE, GB, IT, LI, LU, NL, SE 1. DNA segment of the trp operon from E. coli, characterized by the sequence (coding strand) I 5' CTATCGACC 3' (I) which is connected at the 5' end to the Shine-Dalgarno sequence AAGG, and at the 3' end to the start codon ATG. For the contracting state : AT 1. A process for the preparation of a vector having the trp expression system of E. coli, characterized by cleavage with the restriction enzyme Nco l of an E. coli vector which contains the DNA sequence I 5' GTATCGACC 3' (I) (coding strand) followed by 5' ATGG 3', and a) ligation of a gene structure of DNA sequence II 5' CATG X... 3' 3' Y... 5' (II) in which X and Y denote the first complementary pair of nucleotides downstream of the start codon of a structural gene, in the cleavage site, or b) enzymatic filling in of the cleavage site and ligation of the DNA of the sequence III 5' GTA TCG ACC ATG 3' (III) 3' CAT AGC TGG TAC 5' with a DNA of the sequence IV 5' X... 3' (IV) 3' Y... 5' in which X and Y have the abovementioned meaning, or c) enzymatic degradation of the protruding sequence of the cleavage site, and ligation of the DNA of the sequence VI 5' GTA TCG AC 3' (VI) 3' CAT AGC TG 5' with a DNA of the sequence VII 5' Z ATG X... 3' (VII) 3' Z'TAC Y... 5' Z and Z' denoting any desired pair of nucleotides, which can also be dispensed with.

Description

58994 ■» !?->u This invention relates to modification of the DNA sequence between the Shine-Dalgarno sequence and the start codon of the trp operon to increase protein expression. A Regulation sequences of the trp operon are frequently 5 used for the expression of eukaryotic proteins in E - eoli. A DMA segment containing the promoter and the operator of the trp operon' is now commercially available.

Modifications of the regulation sequences of the trp operon have already been disclosed. Thus,, the possibility 10 of the incorporation of a Hind III cleavage site between the r i bosons I binding site and the start codon in the nucleotide sequence of the regulation element of the trp operon from Serratia narcescens^ for example,, is described in German Offenlegungsschrift 3,247,922 (corresponding to 15 South African Patent No. 83/9519 and Published Australian Patent Application No. 83/22832). The insertion of a CI a I cleavage site into the corresponding sequence of E. coli is disclosed in J.C. Edman et a I., Nature 291 (1981) 503-506. However, this modification alters the number of 20 nucleotides between the ribosomal binding site and the start codon compared with the natural sequence.

It has now been found that a cleavage site for a restriction enzyme can be inserted in the D^A sequence between the ribosomal binding site and the start codon by 25 replacement of a single nucleot ide, that is to say without altering the number of nucleotides- According to the invention, the nucleoside adenosine which is located immediately upstream of the start codon is replaced by cytidine. 30 The invention thus relates to a modified DNA segment of the trp operon of E - coli,,, which is located between the Vi' Shine-Dalgarno sequence and the first start codon and has the DNM sequence X 5s GTATCGACC 3' (I) 3' CATAGCTGG 5s 5 This sequence I is connected at the 5 9 end of the upper strand to the Shine-Dalgarno sequence 5s AAGG 39 3' TTCC 5° of the trp operon (which, at the level of the R N A , corres-10 ponds to the actual ribosoraal binding site), and is connected at the 3° end of the upper strand to the start codon ATG.

The sequence I according to the invention is compared below with the natural E- coli sequence and the sequence 15 disclosed by Edman et a I., op. c i t. , only the upper strand,, called the "coding strand" below, being shown for reasons of clarity: E. coli G i A TCG ACA Edsnan et a I.

GTA TCG AT 20 Sequence I GTA TCG ACC (Alterations from the E. coli sequence are underlined,,) This replacement according to the invention of the A in the natural sequence by C entails the following advantages : 25 If the nucleoside guanosine is connected downstream of the start codon ATG then the recognition sequence CiCATGG 4 for the restriction enzyme Mco I is formed. This cleavage site permits the insertion of DNA in the immediate neighbourhood of the start codon, for example by use of synthetic oligonucleotides of the formula II 5' 3 - CATGX V 3U 5 ' (II) in which X and Y denote the first complementary nucleotide pair downstream of the start codon of a structural gene. in this formula % represents G, and Y represents !0 C, then the Nco I cleavage site is retained in the ligation product. If, in contrast, tor example a synthetic linker is inserted,, whose protruding sequence 5' CATS 3', is connected to another nucleotide, then,, although the Nco I cleavage site is eliminated, on the other hand 15 there is complete variability with regard to the first amino acid downstream of the start codon.

Of course, it is also possible to fill in enzymatica 11 y the protruding ends of the DMA which has been cut with Nco I, for example using Klenow polymerase, and to ligate 20 the blunt-ended DNA of the sequence III,, which has thus been obtained*. 5s GTA TCG ACC ATG 3 3 CAT AGC TGG TAC 5' (III) with a blunt-ended sequence IV 25 (IV) to give the DNA sequence V 5" GTA TCG ACC ATG X ... 38 (V) 3' CAT AGC TGG TAC Y ... 5° 5 x arid y having the abovementioned meanings. No further start codon for a particular structural gene which is to be expressed is required in this. This is favorable tor the preparation of proteins shortened at the N-terminal 5 endfor example™ Another possibility is enzymatic degradation of the protruding ends,, this likewise resulting in a blunt-ended DNA sequence VI 5" GTA TCG AC 3s (VI) 10 3' CAT AGC TG 5' which can be Iigated with a blunt-ended sequence VII 5 s Z ATG X . .. 3" (VII) 3 9 Z'TflC Y ... 5 5 to give the DNA sequence VIII 15 5s GTA TCG ACZ ATG X . . . 3" (VIII) 3' CAT AGC TGZ"TAC Y ... 5" z and Z' denoting any desired nucleotide pair,, which can also be dispensed with. The natural E „ coli sequence is formed when Z and Z* represent A and T respectively. 20 Apart from these diverse cloning possibilities^, the invention offers the advantage that the expression of the structural gene is improved to a surprising extent.

Another aspect of the invention relates to a process for the preparation of a vector containing the trp expression 25 system from E„ coli, which comprises cleavage of an E. coli vector which contains the DNA sequence I (coding strand) followed by 5' ATGG 3" with the restriction enzyme Nco I and © a) ligation of a gene structure of DNA sequence II into the cleavage site or m b) enzymatic filling in of the cleavage site and ligation of the DHA of the sequence 111 with a DNA of / 5 the sequence IV or c) ensyrustic degradation of the protruding sequence of the cleavage site and ligation of the DNA of the sequence VI with the DNA of the sequence VII.

Another aspect of the invention relates to E» coli host 10 organisms which contain a vector obtained according to the invention. Furthermore, the invention relates to a process for the preparation of polypeptides composed of genetically eodable amino acids, which comprises induction., in a known manner,, of expression by E. coli calls 15 which hcive been transformed with a vector according to the invention.

Further aspects of the invent ionf and their preferred embodiments,, are illustrated in detail below and set out in the patent claims- 2o Figures 1 to 3 illustrate the exemplary embodiments in detail- T h u s Figure 1 shows the preparation of the plasmid pH 131/5 from the known pi asm i d pt rpL 1„ Figure 2 shows the preparation of the plasmid pH 185/11 , which codes for interleukin-2, from the known plasmid p 159/6 25 and the plasmid pH 131/5 (Figure 1) „ Finally, Figure 3 shows the plasmid pH 192/5 which codes for /3-interferon.

A particular embodiment of the invention comprises the vector having ampicillin resistance with the /S-lactamase promoter in the same orientation as the trp promoter. 30 This is because it has emerged,, surprisingly, that, owing to the trp operon modified according to the invention, there is not only very pronounced expression of the gene located downstream of the start codon but also the possibility of induction of the ^-lactamase. An increased 7 concentration of ^-lactamase confers resistance to relatively high concentrations of ampicillin, by which means another possibility of selection is opened up.

This makes it possible rapidly to test particularly f a vo-5 rable promoter mutations or modifications of the nucleotides in the region of the ribosomal binding site for each protein which is to be expressed.

If the simultaneous formation of ^-lactamase is un-desired, but the intention is to make use of vectors 10 having ampieillin resistance, it can be suppressed by insertion of a terminator between the structural gene for the desired polypeptide and the structural gene for 0-lactamase. Another embodiment of this aspect of the invention accordingly comprises the insertion of a suitable 15 terminator, preferably a bacterial terminator^ between the abovementioned genes. The terminator of the trp operon is particularly suitable, which is incorporated at a suitable site, for example 10 to 20 nucleotides downstream of the stop codon (or the stop codons) for the 20 structural genep or immediately upstream of the tjS -lactamsse operon.

The gene construct having the trp promoter/operator according to the invention can be incorporated in all plasmids replicating in E. coli. It is advantageous to 25 use the commercially available E. coli vectors, such as pBR 322, pBR 325, pACYC 177, pACYC 184 and pUC 8, and their derivatives. Examples of suitable derivatives are those plasmids from which the non-essent i al regions have been removed, cleavage sites or markers have been intro-30 duced or have been modified.

The gene sequences II, IV, V, VII and VIII conta in, with the nucleotide pair XY, the start of any desired structural gene, for example, in the first place, a gene which is intrinsic to the host and codes for a protein which 35 brings about transport into the periplasmic space or to a 8 cell membrane™ It is possible in this manner to prepare fusion proteins which can be removed from the cytoplasm, and thus be more readily isolated, and/or be protected from degradation by enzymes intrinsic to the cell™ How-5 ever,, it is also possible to generate fusion proteins which,, by reason of their i ns o I ub i I i t y, can readily be separated from the proteins intrinsic to the cell. It is also possible to express the desired proteins directly by placing the structural gene immediately downstream of the 10 start codon A T G „ Examples of polypeptides which can be obtained according to the invention are insulin^ interferons, interleukins, such as interleukin-2, hirudin or somatostatin.

The invention is illustrated in detail by the examples 15 which follow. Reference may be made to the textbook "Holeeular Cloning" by Haniatis et a 1., Cold Spring Harbor (1982), with regard to the individual process steps.

Example 1 Chromosomal E. coli DNA is cleaved with Hinf I, and the 20 492 bp fragment is iniLaosd which contains,, of the trp operons the promoter, operator„ the structural gene of the L-peptide, the attenuator and the codons of the trp E structural gene for the first six amino acids. This fragment is filled in with deoxynucleotide triphosphates by means of 25 Klenow polymerase, connected at both ends to an oligonucleotide which contains a recognition sequence for Hind III, and then cleaved uith Hind HI. The Hind III fragment thus obtained is ligated in the Hind HI cleavage site of pBR 322. The plasmid thus obtained is 30 ptrpE2-1 (J „ C „ Edmann et al., op. c i t.) „ This is transferred as described into the plasmid ptrpLI.

For the conversion of this starting material into a vector according to the invention it is reacted with Cla I 9 as recommended by the aanufecturer (New England Siolabs)™ After incubation is complete the incubation mixture is extracted with phenol, the organic phase is separated off, and the DNA is precipitated by addition of 2 ™ 5 times 5 the volume of ethanol and incubation at -20°C.

The DNA is removed by centrifugation and then treated with alkaline phosphatase (Boehringer Mannheim) in order to remove 5'-phosphate residues.

The synthetically prepared oligonucleotide IX 10 5 * C6ACCAT6GT 3' (IX) is phosphorylated at the 5' end with the enzyme polynucleotide kinase and ATP. For this purpose, the synthetic oligonucleotide is heated at 70°C for 5 minutes and then immediately cooled in an ice bath™ The phosphoryl-15 ation is carried out in 2 5 pi of buffer (50 mM tris.HCl, pH 7.6; 10 mM HgClg, 5 mM dithiothreitol (DTT)) with the addition of 100 juti ATP and about 10 units of ^-polynucleotide kinase, at 37°C over the course of 30 minutes™ The react ion is stopped by addition of the sodium salt of 20 ethylenedianinetetraacetic acid (EDTA) to a final concentration of 50 Wl. Excess ATP can be removed by, for example, gel filtration on sepharose (SEPHADEX * 6 50, fine).

The oligonucleotide IX is self-complementary and can 25 associate with itself to give the double-stranded structure X 5" CGACCATGGT 3" (X) TGGTACCAGC This double-stranded oligonucleotide X has protruding 30 ends which allow insertion into the Cla 1 site of the opened plasmid ptrpLl„ * Trade Mark i 0 About 50 ng of the oligonucleotide are incubated with about 1 pg of the reacted plasmid, which has been treated with phosphatase,, in 30 jjI of buffer (50 mH tris. H CI ^ pH 7-4; f 10 rnH MgC I 210 mM DTT) with the addition of 1 mM ATP 5 and 0*1 ng/ml bovine serum albumin (BSA) at 12°C for 20 hours. The plasmid pH 131/5 is obtained (Figure 1)„ The reaction mixture can be used immediately for the transformation of competent E. coli cells- Selection is carried out on agar plates using L broth (H.J» Miller,, 10 Experiments in Molecular Genetics,, Cold Spring Harborf 1972) and 50 |ug/ml ampicillin.

Since an Nco I cleavage site has been inserted in the plasmid pH 131/5,, the ampi c i 11 in-res istsnt colonies were tested to see whether the plasmid DNA therein contained a 15 Hind 111-Nco X fragment about 300 bp in size. More than 80% of the colonies had this fragment. Sequencing by the Haxam-Gi Ibert method confirmed the incorporation of the synthetic DNA fragment and the sequence of the plasmid pH 131/5 as indicated in Figure 1 „ 20 Example 2 The plasmid p 159/6 as shown in Figure 5 of German Offer)-legungsschrift 3,419,995 is incubated with the enzymes EcoR I and Sal I, as recommended by the manufacturers, and a 420 bp DMA fragment which contains the genetic 25 information for human interleukin~2 is separated off by gel electrophoresis™ The single-stranded protruding ends are degraded with mung bean nuclease (Pharmacia P-L Bio-chemicals) under the conditions recommended by the nanufacturer.

I 30 The plasmid pH 131/5 is reacted with Nco I, and the protruding single-stranded ends are likewise degraded with mung bean nuclease™ This is followed by incorporation of the structural gene for interleukin-2, which is now 11 blunt-ended,., into the plasmid,, which has besn opened and made blunt-ended,, using DNA ligase under "blunt-end" conditions- This results in re-formation of the Nco I cleavage site. Trensformation into E. coli 294 is followed by 5 selection of the ampic i11 in-rss istant clones which have appropriate restriction fragments, for example an Eco R I -Xba I fragment comprising about 260 bp, or an Eco Rl-Sac I fragment having about 150 bp.

The nucleotide sequence was confirmed for the plasmid pH 10 185/11 (Figure 2) by sequencing. The Nco I restriction site is retained in this construct™ For the expression of interIeukin-2, E. coli 294 bacteria which contain the plasmid pH 185/11 are incubated in LB medium (H.J. Miller,, op. cit.) containing 5 0 g / m I ampi-15 cillin, with aeration,, overnight. Then a 1:100 dilution in M 9 medium (H.J,, Miller, op. cit.) containing 1 jug/ml thiamine and 500 jug/ml casamino acids is prepared. At an OD of 0.5,, induction can be carried out with indolyl-3-acrylic acid to a final concentration of 15 ^ig/nl. The 20 bacteria are removed by centrifugation after a further 2 to 3 hours™ It is possible by SDS gel electrophoresis to detect with the induced bacteria a strong protein band which reacts with antibodies against an interleukin-2 prepared in accordance with German Offenlegungsschrift 25 3,419,995. The band corresponds to the expected molecular weight of i nterIeuk in-2 and does not occur with non-induced bacteria. The biological activity of the interleukin-2 can be detected in high concentration in the induced bacteria. 30 The abovementioned conditions for culturing the bacteria apply to shaken flasks™ Higher concentrations of casamino acids and/or L-tryptophan should be added for fermentation to higher OD values (above 3). 1 2 Exam plsj The structural gene for human interferon was obtained from a c0NA bank- The clones contain the inserts in the Pst I site of the plasmid pBR 322. A segment of 120 bp from the (- 5 structural gene of ^-interferon was isolated by exposure to the restriction enzymes Hinf I and Pst I. The recognition sequence of the Hinf I site starts 16 nucleotides downstream of the codon for the N-terminal methionine of this biologically active ^-interferon. 10 The oligonucleotide XI, obtained by synthesis, 5 8 CATGA6CTACAATCTTCTTGG 3 ' (XI) 3* TCGATGTTAGAAGAACCTAA 5' Nco I Hinf I is added onto the Hinf I end of the fragment using DNA 15 Ligase. DNA segment XII is obtained.

Moreovera DNA segment of 365 bp was isolated from the structural gene of - interferon using Pst I and Bg I 11-This segment was cloned in the commercially available plasmid pUC 12 which had previously been reacted with Pst 20 I and Bam HI. The plasmid pH 188 is obtained. After amplification and re-isolation, the plasmid pH 188 is incubated with Pst I and Eco RI, and the *5- interferon gene fragment is isolated (DNA segment XIII).

The plasmid pH 131/5 is reacted with the restriction en-25 symes Nco 1 and Eco RI. This is followed by incubation of DMA segments XII and XIII with the opened plasmid in the presence of the ensyme DNA ligase, under conditions which result in covalent coupling of the linkages. The expected sequence in the plasmid pH 192/5 is confirmed by 30 restriction analysis and sequencing (Figure 3).

The process for the expression of the interferon is 1 3 analogous to Example H. Again, a pronounced band is detected on the electrophoresis gel after induction,, this band not being present with non-induced bacteria. The biological activity of ^-interferon can be detected in the extracts from the bacteria.

The structural gene for interleukin-2 was inserted in the Nco I site of the plasmid pH 131/5 in accordance with Example 2 - After reaction of the plasmid with Eco R J , the protruding ends were made blunt-ended by incubation 10 with Klenow polymerase in the presence of deoxyadenosine triphosphate and deoxythymidine triphosphate.

Into this plasmid which has been opened and made blunt-ended the commercially available terminator of the trp operon (Pharmacia P-L 8iochemica Is) is incorporated under 15 "blunt-end" conditions with simultaneous ring-closure.

After growth and induction of the bacteria as described in Example 2 and previously,,, the bacteria are separated off and Iysed„ SDS gel electrophoresis shows no change in the band of the interleukin-2 protein, which has the 20 same intensity as in Example 2. However, the band corresponding to ^-lactamase exhibits a markedly lower intensity™ I W M

Claims

claims:

1. DNA segment of the trp operon contains the sequence (coding 59 GTATCGACC 3! 5 which is connected at the 5' end sequence A A G G , and at the 3" end from E- coli, which r s t rand) I (I) | r to the Shine-Da Igarno to the start codon ATG.

2. - DNA as claimed in claim 1, which has the sequence (coding strand) la 5' GTATCGACCATGG 39 (la) 10 which is connected at the 5 9 end to the Shine-Dalgarno sequence A A G G , and in which the G at the 3 9 end is the first nucleotide of a structural gene downstream of the start codon.

3. A process for the preparation of a v/ector having the 15 trp expression system of E. coli, which comprises cleavage with the restriction enzyme Nco I of an E„ coli vector which contains the DNA sequence I (coding strand) followed by 59 ATGG 39, and, a) ligation of a gene structure of DNA sequence II 20 5" CATGX ... 3" (II) % G v C a «* i *1 A M in which X and Y denote the first complementary pair of nucleotides downstream of the start codon of a structural gene, in the cleavage site, or 25 b) enzymatic filling in of the cleavage site and ligation of the DNA of the sequence III 5* GTA TCG ACC ATG 3' (III) 3' CAT AGC TGG TAC 5" with a DNA of the sequence IV 30 59 X ... 39 (IV) 3 9 Y ... 5 9 in which X and Y have the abovementioned meaning, or c) enzymatic degradation of the protruding sequence of the cleavage site, and ligation of the DNA of the 35 sequence VI J 1 5 5* GTA TCG AC 3e (VI) 3® CAT AGC TG S* with a DNA of the sequence VII 5' 2 ATG X ... 3' (VXD 5 3" Z'TAC Y ... 5' Z snd 10 denoting any desired pair of nucleotides^ which can also be dispensed with.

4. E . coli which contains a vector obtained according to claim 3, Z representing C and Z' representing G. 10

5. A process for the preparation of a polypeptide composed of genetically codable amino acids, which comprises induction of expression of E. coli cells as claimed in claim 4 .

6. Plasmid pH 131/5 as shown in Figure 1. 15

7. Plasmid pH 185/11 as shown in Figure 2.

8. A DNA segment according to Claim 1, substantially as hereinbefore described and exemplified*

9. A process according to Claim 5 for the preparation of a polypeptide composed of genetically codable amino acids 20 substantially as hereinbefore described and exemplified.

10. A polypeptide composed of genetically codable amino acids, whenever prepared by a process claimed in a preceding claim. F. R. KELLY & CO,, AGENTS FOR THE APPLICANTS. )