Synthesis of a human insulin gene VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage

201

Gene, 27 (1984) 201-211 Elsevier GENE

954

Synthesis of a human insulin gene VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in Ml3 bacteriophage + (Recombinant DNA; affinity leader sequence; C-peptide; vector plasmids; lac promoter; Bgalactosidase; radioimmune assay)

F. Georges, R. Brousseau, J. Michniewicz, G. Prefontaine, J. Stawinski, W. Sung, R. Wu * and S.A. Narang ** Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KlA OR6 (Canada) Tel. (613) 996-3985, and *Section of Biochemistry, Molecular and Cell Biology, Cornell University,Ithaca, NY 14853 (U.S.A.) Tel. (607) 256-2205 (Received

July 5th, 1983)

(Revision

received

(Accepted

October

October

8th, 1983)

12th, 1983)

SUMMARY

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M13mp8 vector. A clone pNB82-121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25bp afIimity leader sequence coding for (glu), was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of pgalactosidase. Preproinsulin was expressed under Zac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide. -

INTRODUCTION + Dedicated

to the memory

of Ahmad

** To whom all correspondence

I. Bukhari.

and reprint

requests

should be

addressed. Abbreviations: threitol;

bp, base pairs;

EtBr, ethidium

actopyranoside;

pfu, plaque-forming

RIA, radioimmune

leader bottom

leader

SL-IIBS

SSC,

pH 7.6; TE buffer, triethylammonium

0378-I 119/84/$03.00

strand;

15 mM

10 mm Tris-I mm EDTA bicarbonate;

0

TLC,

1984 Elsevier

(bottom),

sub-

(top), subassembly

subassembly

NaCl,

form;

sulfate; SL-ITS

SL-IBS

SL-IITS

(bottom),

150mM

DTT, dithio-

isopropyl-PD-thiogal-

units; RF, replicating

leader I top strand; I bottom

II top strand; strand;

IPTG,

assay; SDS, sodium dodecyl

(top), subassembly assembly

BCA, proinsulin;

bromide;

Recent advances in the rapid chemical synthesis of deoxypolynucleotides offer an attractive approach for designing and constructing DNA sequences for biological studies. Such an approach gives flexibility in the selection of codons to introduce various restriction sites, exclusion of intron sequences and to

buffer;

thin-layer

Science

leader II

Naa’citrate, TEAB,

chromato-

Publishers

graphy;

X-gal, 5-bromo-4-chloro-3-indolyl-/?-o-galactoside;

polymerase

500 mM NaCl; YT medium, extract

10 x

buffer, 70 mM Tris . HCl pH 7.5 - 70 mM MgCl, -

5gjl - NaCl 5g/l.

Bacto tryptone

Sgjl- Bacto yeast

313

limit the length of DNA function

to examine

of a given sequence.

the synthesis (Brousseau describe

the biological

Previously

and cloning of human proinsulin

DNA

et al., 1982). In this communication,

(i) the assembly

we

proinsulin

DNA;

preproinsulin

gene with an affinity leader sequence, expression

(iii) linking studies

tutions

at four different

expressed vectors

of the

using

these substi-

do not alter the genes have been

specially

strong promoters &)

constructed such as trpEp,

promoter

/1. The level of expression

of bac-

may also be

by using a vector with a high copy number.

In this communication single-stranded DNA

a modified

we report the use of small bacteriophage M 13mp8 vec-

tor for cloning, sequencing, site-specific mutagenesis and expression studies of synthetic human preproinsulin gene. This vector already has a fat promoter and several useful restriction sites in the fl-galactosidase gene, and the synthesis of the fused gene pro-

replaced with glycine, 5 - methionine with isoleucine, 11 - leucine with isoleucine, and 15 - alanine with isoleucine. These changes were introduced to in-

duct is inducible. The main advantages of this vector are (i) its high copy number of 200-300 molecules per bacterium (Messing et al., 198 1); (ii) its suitability for

crease the number of restriction sites, such as four Sau3A and two BamHI sites in the codon region Pre-peptide

positions

and the leftward

increased

pre sequence of human proinsulin (Goeddel et al., 1980) as shown in Fig. 1. The present sequence contains four substitutions, at positions 2 - alanine

____

containing

of the pre se-

Moreover,

of the pre sequence.

in bacteria

teriophage

in the Ml3

studies we synthesized

property

lacZpUV5,

system.

In the present

study.

To date. most of the eukaryotic

gene; (ii) site-specific

of pre- and

bacteriophage

for detailed

hydrophobic

to remove an EcoRI site at the junction

and (iv) its initial

quence

of a leader (pre) sequence

and its joining to the proinsulin mutagenesis

which will allow further modification

we reported

__

12 3 4 5 6 7 a 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 MET GLY LEU TRP ILE ARG LEU LEU PRO LEU ILE ALA LEU LEU ILE LEU TRP GLY PRO ASP PRO ALA ALA ALA GUI PHE ARG MET PHE VAL ASN GLN ?b?Yi .&le,Tir PJll'iI Szu3A tihd Sau3A AvaII BmlHI EocPI ---_ ._-____ -----_-____ __-- --_ I6 ~5-AATTcATG GGC CTA TGG ATC CGT CTA CTG CCT CTG ATC'GCG CTG CTG ATC'CTC TGG GGA CCG'GAT CCA GCT GCG GCC dsA TTC LGG ATG TTT GTC AAT CAG ,TAC CCG GAT ACC TAP GCA GAT GAC GGA,GAc L12

TAG CGC GAC,GAC

Lll

TAG GAG ACC CCT GGC CTA GGT GGA CGC CGG CTT AAG GCC TACIAAA

LlO

---

L9

L8

L7

_

B-Chain

CAG TTA GTC

~____

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 59 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 HIS LEU CYS GLY SER HIS LEU VAL GLU ALA LEU TYR LEU VAL CYS GLY GLU ARG GLY PHE PHE TYR THR PRO LYS THR ARG ARG GLU ALA GLU ASP

CAC CTT TGT GGT TCT CAC CTG GTG GAG GCT CTG TAC CTG GTG

GAA GCT GAA Gk

GTG GAA ACA CCA AGA GTG GAC CAC CTC CGA GAC ATG GAC CAC

CTT CGA CTT CTG

_______

C_pepti,je ~__.__~____~~~~~~~.

~~~ ~~~~~~~~~~

.

65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 LEU GLN VAL GLY GLN VAL GLU LEU GLY GLY GLY PRO GLY ALA GLY SER LEU GLN PRO LEU ALA LEu GLU GLY SER LEU GLN LYE ARG GLY ILE VAL _ _J&'i CTT CAA GTG GGT CAA GTT G4A CTT GGT GGG GGT CCT GGT GCG GGT TCT CTT CAA CCT TTG GCT CTC GAG GGA TCA CTT GAA AAG GAA GTT CAC CCA GTT cm

CTT GM

CGT GGC ATT GTG

CCA ccc CCA GGA CCA CGC CcA AGA GAA GTT GGA AAC CGA GAG CTC CCT AGT GAA GTT TTC GCA CCG TAA CAC -1 L-

A-Chain

__

97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 GLU GLN CYS CYS THR SER ILE CYS SER LEU TYR GLN LEU GLU ASN TYR CYS ASH TEp

GAG CAG TGC TGC ACC AGC ATC TGC TCC CTC TAC CAA CTG GAG AAC TAC TGC AAC TGA G CTC GTC ACG ACG TGG TCG TAG ACG AGG GAG ATG GTT GAC CTC TTG ATG ACG TTG ACT CCTAG-5' _',.. ,,, Fig. 1.

Human preproinsuhn

sequence.

coding

sequence.

The amino acid 1 to 24 represents

with glycine;

5 - methionine

Ll to L12 represent

the leader (pre) sequence

with isoleucine;

the synthetic which contains

11 - leucine with isoleucine;

oligonucleotide

fragments

four substitutions:

and 15 - alanine

comprising

the

pre DNA

position 2 - alanine was replaced

with isoleucine.

6215

.

1 Met Thr

pNBa2-

121 : 5‘-iTG.

Met

Ile

Thr 1

ACC. ATG. ATT. ACG:AA.TTC.

N-Terminal +

Reproineulin

ATG.GGC

. . . . . . . . . . . . . . . . . GCT. GCG.GCC.GA

DNA +

I ATT A CGC CGG CT-G

I Preproinsulin Met pNl363-

26

: 5:ATG.

Thr

Met

Ile

Renlovol C CGG. ATG.TTT. . . . . . . . . . . TGA.GkATCC G CC TAG AAA p- 5’ Primer I

Removal of ATT triplet by&e-specific mutogenasis

DNA (- ATT)

Thr

ACC. ATG. ATT

1 ACG. AA. TTC

ATG. GGC . . . . . . . . . . . . . . . . ... GCT. GCG.GCC.

GAC.

CGGATGTT

1 .. . . . . . . . . . . . . . . . . . . . . . . . . TGA .GGATCC

Eco RI- siW I. EcoR-I digest of RFform Insertion of Affinity Leodu.

TGT.TGT.TGT. ‘XACA.ACA.

TGT.TGT.TGT. TGT.G ACA.ACA.ACA.ACA.C.TTAAp-5’ Affinity

Met

Thr

Met

Ile

Thr

I

Asn

Cys

pNsa3 - ia : 5)-ATG. ACC. ATG. ATT. ACG. AAT.TGT.

Cys

Cysteine Cys Cys

Leader Cys Cys Cys

TGT.TGT.TGT.TGT.TGT.TGT.

Asn pNsa3

-I~:~ATG.A~~.ATG.ATT.

A~,G.

Ser

Gln

Glutamine Gin GInGIn

LeaderGln

Phe Get

Repminsulin

1 C.Termirml )

4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TGA.GGATCC

Eco R-Isire Reproinsulin Gln

AAT. T~A.CAA.CAA.CAA.CAACAA.CAA.TTCATG Eco RI

N-Teninol Glu

GAA.TTC.ATG

I -

Leoder

Phe

C-Toq$l

4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TGA.GGTTcc

II

Fig. 2. Construction of various Ml3 vectors. Clone pNB82-121 is human preproinsulin DNA inserted in M13mp8; pNB83-26 was derived from pNB82-121 by (Am) triplet deletion using synthetic primer; I, pNS83-18 contains seven-cysteine codon produced by inserting a synthetic 25-mer; II, pNS83-16 is the complementary strand containing seven-glutamine codon.

site-specific mutagenesis followed by dideoxy sequencing to identify the mutants; (iii) its ability to produce reasonable levels of a eukaryotic protein such as interferon (Slocombe et al., 1982). To simplify isolation and purification of a pure protein from genetically engineered bacteria, we thought of attaching a short stretch of homopolypeptide sequence before the pre sequence. For example polycysteine, polyglutamine, polylysine, polytryptophane or polyleucine could change the physical property of the desired product so that its isolation from the complex mixture of the cell protein may become easier by affinity column chromatography. The details of this approach will be published elsewhere. In the present paper we describe the DNA synthesis to introduce TGT codon of (cysteine), in one strand and CAA codon (glutamine), on the opposite strand preceding the pre sequence as outlined in Fig. 2. This aflinity leader sequence put preproinsulin gene in phase with the B-galactosidase AUG intiation codon. In the case of (glutamine), leader, an expression was observed whereas with seven cysteine leader the expression was undetectable. Whether this was due to rapid degradation caused by the

cysteine leader, interference of the cysteine with the immunoassay system or some other reason will be investigated.

MATERIALS AND METHODS

(a) General method of chemical synthesis of deoxyribooligonucleotides The chemical synthesis of deoxyribooligonucleotides was carried out by the modified phosphotriester method in solution, with some moditications (Narang et al., 1979) described below. (I) Phosphorylation reaction 1,2,4-Triazole (4.1 molar equivalents) was added to 5 ’ -dimethoxytrityl N-protected deoxyribonucleotide (1 molar equivalent) in pyridine solution at room temperature. p-Chlorophenyl phosphodichloridate (2 molar equivalents) was then added and the reaction mixture was stirred for 5 min to complete

phosphorylation

reaction

sis. P-Cyanoethanol

as checked by TLC analy-

(9 molar equivalents)

added and the reaction

allowed to proceed

3 h at room temperature. the fully protected fied on reversed matography

another

After the usual work-up,

deoxymononucleotide phase

was next

(RP-2)

as described

was puri-

short column

earlier

(Hsiung

chroet al.,

(1: 10, 1: 100 and 1: 1000) was used to transfect petent

HBlOl

cells. More

were obtained

on ampicillin

selected at random

fragment

dNTPs,

(2) Coupling reaction

hardt solution,

[ r”-P]dATP

ization at 52-55°C Before each coupling

reaction

the b-cyanoethanol component

was re-

moved by treatment with diisopropylamine (Sung et al., 1982) in pyridine solution at room temperature for 15 min. After evaporating in vacua, the residual material was coupled with 5’-hydroxyl component in a minimum amount of pyridine in the presence of mesitylene sulfonyl tetrazole for 15 min at room temperature. The reaction mixture was diluted with dichloromethane and washed with 2 “/b sodium bicarbonate. After evaporation of solvent from organic extract, the residue was purified by preparative silica gel TLC 50%-water

(solvent dioxane 40:‘,-ethyl acetate 10%) for both 17- and 25-mers.

(b) Assembly of pre DNA sequence and its joining to BCA proinsulin gene in vector PSI-BCA4 Oligomers up to 37 bases long were assembled as described previously (Brousseau et al., 1982). Approx. 5 pmol of each of the complementary strands SL-ITS (top) (5’-AATTCATGGGCCTATGGATCCGTCTACTGCCTCTGATC) (38-mer), (bottom)(5’-CAGCGCGATCAGAGGCAGTAG-

SL-IBS

ACGGATCCATAGGCCCATG) (40-mer), SLIITS (top) (5’-GCGCTGCTGATCCTCTGGGGACCGGATCCAGCTGCGGCCG) (40-mer), SLIIBS (bottom) (5’-AATTCGGCCGCAGCTGGATCCGGTCCCCAGAGGATCAG) (38-mer) were dissolved in 10 ~1 of 0.5 M Tris . HCl (pH 8.0) and annealed by heating to 95” C followed by slow cooling overnight in a water bath. To this was added 0.17 pg (67 fmol) of EcoRI digested plasmid PSIBCA4 (Brousseau et al., 1982), 2 ~1 each of 0.1 M MgCl,, 0.1 M KCl, 30 mM DTT, 4 mM ATP. The volume was brought to 19 ~1 with water and 1 ~1 of T4 polynucleotide kinase was added. After 1 h at 37°C 1~1 of TbDNA ligase was added and the sample was kept overnight at 14°C. A total of 12 ~1 of the sample in three dilutions

plates. 50 colonies were

cellulose filters for colony hybridization. as a template,

clones.

on nitroA 30-base-

using SL-ITS

oligomer

L-10 as a primer (see Fig. 1). and Klenow enzyme. Hybrid-

(2 x SSC, 0.5”,

2 mM EDTA)

Digestion

pNB82-153

com-

300 transfectants

and grown in duplicate

long probe was constructed

1980).

group from the fully protected

than

of plasmid

with EcoRI

SDS, 1 x Den-

revealed two positive

showed

DNA a band

pected length (74 bp) on 8”, acrylamide

from

clone

of the exgel electro-

phoresis. (c) Cloning of preproinsulin

DNA in M13mp8

PSI-BCA4 plasmid DNA (10 pg) was digested for 1 h at 37°C with 10 units of EcoRI and 10 units of BamHI. Similarly, 10 pg of pNB82-153 plasmid DNA was digested for 1 h at 37°C with 10 units of EcoRI enzyme. The reaction mixtures were heated at 65 ‘C for 10 min to denature the restriction enzymes, the DNA was precipitated, redissolved in TE buffer (10 ~11)and applied to a 5 O0 acrylamide (1:20) TB buffer-gel which was run overnight at 70 V. pBR322 DNA digested by Alla11 was used as a marker. After staining the gel with EtBr, the bands corresponding to the proinsulin DNA from PSI-BCA4 and the pre sequence from pNB82-153 were cut out of the gel, crushed and extracted three times with a total of 30 ml 0.1 M TEAB. The DNA from each solution was concentrated by passing through a microcolumn containing 200 ~1 of DE52 resin; the DNA was eluted out of the resin by washing with 5 x 25 ~1 3 M Tris . HCl (pH 8.0). It was next diluted with 2 ~01s. of water and 1.2 ml of ethanol. After cooling at -70” C for 15 min, the precipitated DNA was isolated by centrifugation. Approx. 125 ng of each of these two eluted DNAs were ligated with 40 ng of M 13mp8 (RF form) which had been cut previously with EcoRI and BamHI. The ligation volume was 12 id and the reaction was performed for 6 h at 12 ’ C. The resulting mixture was used to transfect competent JM 103 cells. After plating on X-gal/IPTG-containing plates, 23 transfectants were obtained. The DNA sequence of one such clone, pNB82-121, was determined by recloning the larger EcoRI-BamHI insert in M13mp9 and found to

ACGT

cc TCA c

Tf G@ TT

TGT

CC CA G TCA

GTCAA TT GT et. T [: GGA c TTC tB G [ AA CiG cTG C

G CA

TC A

GG

cc A

G

GG G

AC

G T

TG TC CC rG c AT

A

COT !

G G

3 F 7

Ma

A

I

; A

EC RI

site

G

Fig. 3. Autoradiogram of leader sequence and of the starting sequence of preproinsulin in M13mp9 by dideoxy method 6showing two EcoRI sites.

+------

8 -chain

L

. Leader

sequenca

207

have the correct sequence by the dideoxy method as shown in Fig. 3. (d) Removal of EcoRI site between pre- and proinsulin DNA in pNB82-121 vector through triplet depletion A 19-base-long primer (S-AAACATCCGGTCGGCCGCA) corresponding to the desired deletion was synthesized and purified by gel electrophoresis as described above. 200 pmol of this purified material were phosphorylated using T4-polynucleotide kinase and ATP. 20 pmol(3 $1) of this phosphorylated 19-mer were mixed with approx. 1 pmol(2 ~1)of a single-stranded form of phage NB82-121. 2 ~1 of 10 x polymerase buffer, 1~1 of 0.1 M DTT and 2 ~1 of H,O were added and the mixture was annealed by heating at 65 ‘C for 3 min and then chilling in ice. Next were added 0.4 ~1of each dNTP 10 mM, 0.5 ~1of 10 mM ATP, 0.5 ~1of Klenow fragment (1 unit) and 0.25 ~1 of T6DNA ligase. The reaction was allowed to proceed for 24 h at 12°C. Diluted aliquots of the reaction mixture were used directly to transfect JM103 log-phase cells. Approx. 2000 plaques were obtained. 46 plaques picked at random were transferred onto a nitrocellulose filter together with two negative controls [JM103/mpB (without preproinsulin DNA) and JM103/NB8212 1 (with preproinsulin DNA)]. These colonies were grown on YT plates, then lysed and denatured as per the procedure of Grunstein and Hogness (1975). Colony hybridization was accomplished with a kinased 1Pmer probe (1.2 x lo6 cpm) in 6 x SSC, 1% dextran sulfate, 0.05 % Triton X-100, overnight at room temperature. Washing the filters at progressively increasing temperature up to 58°C (20 ml 6 x SSC, 0.05 % Triton X-100 each time) gave two clones hybridizing strongly at 58°C while all other clones and the two controls has disappeared. One of these clones, pNB83-26, was sequenced by the dideoxy method and found to have undergone the desired deletion as shown in Fig. 4. (e) Insertion of synthetic affinity leader sequence at EcoRI site of vector pNB83-26

was formed by annealing 15 pmol of each of two phosphorylated 25-mer in 4 ~1 of 20 mM Tris * HCl (pH 7.5) and 0.1 mM EDTA at 65°C for 3 min. The annealed duplex (0.5 ~1) was mixed with 1 pg of EcoRI digested RF form of pNB83-26 in 4 ~1 of 100 mM Tris * HCl (pH 7.5), 10 mM MgCl,, 0.1 mATP and T6DNA ligase. The solution was incubated for 3 h at 15 ’ C. After addition of 0.2 M EDTA (2 ~1)and H,O (23 ~1)the solution was used to transfeet JM103 cells as described above, and about 150 plaques were obtained using 3% of the ligase reaction mixture. (f) Isolation and characterization of phages containing each strand of affinity leader sequence Phages were picked and grown for 5 h in 2YT solution with JM103 hosts at log phase. After centrifuging at 5000 rev./min for 10 min, the bacteriophage solution (40 ~1) was treated with 2.5 M NaOH (10 ~1) for 10 min and then diluted with 1 M Tris * HCl (pH 7.0), 1 M NaCl and 50 m&I EDTA. The phage DNA solution was applied to the two sheets of nitrocellulose filter paper with one as duplicate. After heating at 80’ C under vacua for 2 h, both filter papers were washed with 30 ml (6 x SSC and 0.05 % Triton X-100) solution. They were next treated with a prehybridization mixture of 6 x SSC, 1y0 dextran sulfate, 1 x Denhardt solution and 0.05% Triton X-100 for 1 h. The filter papers were rinsed again by 10 ml of 6 x SSC and 0.05% Triton X-100. For hybridization, the filter papers were put into a sealed cooking bag containing 5 ml of 6 x S SC, 1y0 dextran sulfate, 0.05% Triton X-100 and lo6 cpm of the appropriate probe such as (i) 32P-AATTGTTGTTGTTGTTGTTGTTGTTGTG, or (ii) 32P-AATTCACCAACAACAACAACAACAAC. After 16 h, the filter papers were washed at room temperature with 20ml of 6 x SSC and 0.05% Triton X-100. Autoradiography of the two filter papers indicated 10 out of 80 plaques were positive for probe (ii), 5 plaques positive for probe (i). Single-stranded DNA was prepared by the usual procedure and the positive clones were confirmed for its sequence by the dideoxy method (Fig. 5).

AduplexofS’pAATTGTTGTTGTTGTTGTTGTTGTG CAACAACAACAACAACAACACTTAAp-5’

20x

pNS83-18

pNS83-16

ACGT

ACGT

TAC

TGG TAC

.aw e

TAA TGC

0

TT %

ii TT

TC) TTG G

TT TF c TT G TT A A G

e a

;: C

l

:

!i

G

0 0

G G

G A

0

1 A

o

II

* 0

[e T

0

Fig. 5. Autoradiograms panel)

sequenced

T A C C

of seven-cysteine-codon

by the dideoxy

method.

region in pNS83-18

(left panel) and of seven-glutamine

T

codon region in pNS83-16

(right

(g) Induction of preproinsulin synthesis in Ml3 system Overnight grown culture of E. coli (JM103) was diluted by 2YT medium with 0.2% glucose in the ratio of 1: 100. After growing for 1 h at 37 “C, it was infected with the appropriate phage at 100 pfu/cell. IPTG was added to a final concentration of 0.7 mM. Aliquots (8 ml) were taken at different intervals and cells were collected by centrifugation at 5000 rev./ min for 10 min. The supernatant was stored for radioimmunoassay and the cell pellet was suspended in 2 ml of 6 M guanidine * HCl phosphate buffer. After lysing the cells by sonication, the cell debris was removed by centrifugation at 10000 rev./min for 20 min and the cell content solution was analyzed by radioimmunoassay for C-peptide as described below.

====5=>_

TIME

Fig. 6. Production infection

18 (cysteine

After cell growth, both culture media and cell content were assayed for human preproinsulin by human [ 1251]tyrosine C-peptide antibody kit (Novo Industri) with synthetic human C-peptide as a standard.

IO

I

20

30

FOR IPTG

was measured series);

INDUCTION

(h)

by E. coli K-12 (JM103) after

of preproinsulin

peptide. Circles, pNS83-16

a

___r===:==

I

with clone pNS83-16

preproinsulin

(h) Radioimmunoassay for C-peptide

TABLE

___

I

or pNS83-18.

The activity

by radioimmunoassay (glutamine

squares,

against

series); triangles,

clone pNS83-31.

Closed

with solid line: cell pellet; open symbols with broken

of C-

pNS83symbols

line: super-

natant.

I

Production

of hybrid

Affinity

preproinsulin

by M13mp8 Estimated

Clone

preproinsulin

yield a

(ng/ml culture)

leader

Time after IPTG induction 2

(h)

4

10

21

21

51

Whole-cell sonicate -gln-

pNS83-16

103(95)

-gln-

pNS83-53

96(92)

-cys-

pNS83-18

b

<20’

<20”

-cys-

pNS83-31

d

<20”

<20’

-gln-

pNS83-16

30

70

138

216(218)

258

216

-gln-

pNS83-53

35

65

90

235(220)

282

237

-cys-

pNS83-18’

<20’

<20’

-cys-

pNS83-3 1 d

<20’

<20’

125

150(118) 165(128)

Supernatant

a Yield estimated

by C-peptide

b Other clones pNS83-21,25,29, in C-peptide

radioimmunoassay.

c This value is actually d Clone pNS83-31,

radioimmunoassay, 37,44, Results

indistinguishable

as control,

60,66,

except those in brackets 71 and 74, containing

of pNS83-37,

by insulin radioimmunoassay.

the same affinity leader sequence,

66 and 74 were also negative

from the background,

gave similar negative

in the insulin radioimmunoassay

as evident in the case of the control.

has three copies of the TGT 25-mer and is therefore

out of phase in M13mp8

vector.

test.

results

lar C-peptide the amount

toward

basic assumption preproinsulin proinsulin. marized

antiserum

of preproinsulin

(Faber

et al., 1978)

was estimated

on the

that both the target protein

have the same radioimmune

and

activity as

The results are shown in Fig. 6 and sum-

with a deletion

of the AAT triplet from the sequenc-

ing strand (Fig. 4). This was also confirmed the EcoRI-Hind111 sequence

fragment containing

of preproinsulin

DNA on a 4’ o polyacryl-

amide gel (not shown).

in Table I. (d) Insertion of affinity-leader preproinsulin

RESULTS

by sizing

the complete

AND

The production

DISCUSSION

DNA methodology stability

(a) Chemical synthesis The chemical synthesis of fragments was carried out by modified phosphotriester method (Narang et al., 1979). For longer fragments (17-25mer) serious streaking phenomena arise on silica gel; these could be overcome by using a dioxane-ethyl acetate-water solvent system. Each intermediate was purified by preparative TLC. The overall time taken for five individual coupling reactions is approx. 6 h. (h) Enzymatic

assembly of synthetic

fragments

ic fragments (Ll to L12) in the presence of non-radioactive complementary strand using T4-DNA ligase as described earlier (Brousseau et al., 1982). This was then joined to EcoRI-digested plasmid PSIBCA4 and the mixture was used to transfect competent cells. Two positive clones were identified by colony hybridization and were recloned in M13mp8. For sequence

analysis,

clone pNB82-121

was par-

tially digested with EcoRI and BamHI. The larger fragment was recloned in M13mp9 which was convenient for sequencing because of the proximity of the universal primer available to EcoRI sites. (c) Site-specific

mutagenesis

Since we intended to study the expression of preproinsulin in various systems besides M 13 it was desirable to remove the EcoRI site between pre region and proinsulin. It was achieved by site-specific mutagenesis deleting an ATT triplet using a synthetic 1Pmer primer as outlined in Fig. 2. Selection of the clones by colony hybridization at various temperatures up to 58°C gave two positive clones, one

of a peptide

using recombinant-

poses two serious problems:

of a peptide,

(ii) tedious

method

(i)

of purili-

cation. Regarding (i) the desired peptide can be stabilized by fusing with another peptide. Regarding (ii) it could be overcome by inserting a homopeptide leader sequence at the end of a desired peptide. This leader would serve as an affinity handle to purify the desired peptide. In the present studies we inserted (cysteine), or (glutamine), the preproinsulin DNA

residues as a leader before in M13mp8. This was

achieved by inserting synthetic 25mer duplex as outlined in Fig. 2 and shown in Fig. 5 by DNA sequence analysis. Positive clones were checked for the expression

The 72-bp pre sequence flanked by EcoRI termini was assembled by stepwise joining of twelve synthet-

sequence upstream of

gene

of preproinsulin.

(e) Production

of preproinsulin

in M13mp8

IPTG induction of cultures of E. coli K-12 (JM103) infected with M13mp8 clones pNS83-16 or pNS83-18 resulted in a reasonable level of expression of preproinsulin in the supernatant (282 ng/ml culture) and bacterial lysate (165 ng/ml culture) only in the case ofglutamine heptamer leader (pNS83-16). The cysteine leader (pNS83-18) gave a negative result, as shown in Table I and Fig. 6. The presence of preproinsulin was checked by its positive signal for human C-peptide as well as A-B-peptide in the RIA tests which is approx. loo,/, on a molar basis. The data in Fig. 6 represent the corrected value. The preproinsulin activity in the bacterial lysate increased rapidly after induction with IPTG, reaching maximum at about 3 h and remaining fairly constant up to 21 h. The activity increased more slowly in the bacterial supernatant than the cell lysate, but reached a value three times higher after 21 h, indicating that preproinsulin is released slowly from the infected bacteria. Whether this release is due to secretion or leakage is presently unknown; further work is in progress in this direction.

211

As shown in Fig, 6, the clone pNS83-18 containing seven-cysteine leader failed to show any crossactivity with human C-peptide or A-B-peptide RIA assay, whereas clone pNS83-16 containing sevenglutamine leader gave a positive response. Although both recombinant plasmids possessed the preproinsulin sequence in the same reading frame as that of j?-galactosidase amino terminus, the failure could be attributed to a quick proteolysis of the translated product. A similar observation has also been reported in the case of metallothionein hybrid protein (Thirion, J., Mbikay, M. and Brzezinski, R., personal communication). Mbikay et al. (1983) and Murakami et al. (1979) have already shown that some abnormal proteins are quickly digested in E. coli as preferential substrate for bacterial proteases. The failure of expression could also be due to the inability of the cellular system to translate messenger RNA containing specific repeat sequence because of its unique secondary structure or severe disruption of the preproinsulin secondary structure by the stretch of seven cysteine which would make the C-peptide immunoassay inoperative. This interesting observation is under further investigation.

ACKNOWLEDGEMENTS

We thank Drs. D.Y. Thomas and P.P. Stepien for assisting us in the RIA test. NRCC No. 22762.

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Faber, O.K., Binder, C., Markussen, J., Heding, L.G., Naithani, V.K., Kuzuya, H., Blix, P., Horwitz, D.L. and Rubenstein, A.H.: Characterization of seven C-peptide antisera. Diabetics 27, Suppl. 1 (1978) 170-177. Goeddel, D.V., Gray, A. and Ullrich, A.: Nucleotide sequence of human preproinsulin complementary DNA. Science 208 (1980) 57-59. Grunstein, M. and Hogness, D.S.: Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72 (1975) 3961-3965. Hsiung, H.M., Brousseau, R., Michniewicz, J. and Narang, S.A.: Synthesis of human insulin gene, 1. Development of reverse phase chromatography in the modified triester method. Its application in the rapid and efftcient synthesis of eight deoxyribooligonucleotides fragments constituting human insulin A-chain DNA. Nucl. Acids Res. 6 (1979) 1371-1385. Hsiung, H.M., Sung, W., Brousseau, R., Wu, R. and Narang, S.A.: Synthesis of the human insulin gene, III. Chemical synthesis of 5’-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA. Nucl. Acids Res. 8 (1980) 5753-5765. Mbikay, M., Brzezinski, R. and Thirion, J.: Differential expression of cloned mouse metallothionein sequences in Escherichiu coli. DNA 2 (1983) 23-30. Messing, J., Crea, R. and Seeburg, P.H.: A system for shotgun sequencing. Nucl. Acids Res. 9 (1981) 309-321. Murakami, K., Voellmy, R. and Goldberg, A.L.: Protein degradation stimulated by ATP in extracts ofEscherichiu coli. J. Biol. Chem. 254 (1979) 8194-8200. Narang, S.A., Hsiung, H.M. and Brousseau, R.: Improved phosphotriester method for the synthesis of gene fragments, in Wu, R. (Ed.), Methods in Enzymology, Vol. 68, Recombinant DNA. Academic Press, New York, 1979, pp. 90-109. Slocombe, P., Easton, A., Boseley, P. and Burke, D.C.: High-level expression of an interferon a2 gene cloned in phage M13mp7 and subsequent purification with a monoclonal antibody. Proc. Natl. Acad. Sci. USA 79 (1982) 5455-5459. Sung, W. and Narang, S.A.: Modified phosphotriester method for chemical synthesis of ribooligonucleotides, Part I. Synthesis of undecadenylate and two fragments constituting the sequence of R-17 translational control signal. Can. J. Chem. 60 (1982) 11l-120. Communicated by AI. Bukhari.