Recombinant DNA means and method

Living cells containing genetic material derived from recombinant DNA material and capable of expressing rennin, pre-prorennin and prorennin. The rennin, pre-prorennin and prorennin are derived from cells which are themselves or have had parents thereof treated by recombinant DNA methods to allow production of the desired enzymatic proteins during growth in culture.

BACKGROUND OF THE INVENTION 
The enzymatic protein rennin has long been known as useful for coagulating 
milk casein in cheese making. It is also used in connection with 
cheese-ripening because of its specific proteolytic activity. In the past, 
it has been obtained from rennet in commercial manufacture. Milk-fed 
calves can be butchered and the fourth stomach removed freed of its food 
content. A complicated method is then used wherein the stomachs are dried, 
salted, and frozen. At factory points, the stomachs are washed, freed of 
salt and treated to remove surface fat. They are then stretched on racks 
and dried. The dried stomachs are often cold stored then ground and placed 
into large vats with a brine solution circulated through the skins until 
extraction of rennin is completed. The above procedure for preparing 
rennin is costly, and presents many difficulties in producing large 
amounts needed for commercial use in various applications throughout the 
world. 
SUMMARY OF THE INVENTION 
It is an object of this invention to obtain living cells which are capable 
of producing rennin in culture for volume production. 
It is a still further object of this invention to obtain living cells which 
are capable of producing prorennin in culture for volume production. 
It is a still further object of this invention to obtain living cells which 
are capable of producing pre-prorennin in culture for volume production. 
It is another object of this invention to provide rennin, prorennin, or 
pre-prorennin derived from living cells in accordance with the preceding 
objects which living cells contain genetic material derived from 
recombinant DNA material. 
It is still another object of this invention to provide specialized rennin 
genes, pre-prorennin genes and prorennin genes. 
It is still another object of this invention to provide methods of 
producing rennin, prorennin or pre-prorennin using recombinant DNA 
techniques. 
It is still another object of this invention to provide signal sequences 
for use in transporting selected amino acid sequences such as selected 
enzymes or protein material to periplasmic space, other cellular areas or 
extracellularly with the appropriate host. 
It is still another object of this invention to provide particular modified 
cells for use in production of polypeptides displaying rennin or milk 
clotting activity. 
According to the invention, living cells contain genetic material derived 
from recombinant DNA material and are capable of expressing rennin or 
pre-prorennin or prorennin. The invention also comprises the rennin, 
prorennin and pre-prorennin and the genes therefor, derived from living 
cells. 
According to a method of this invention, expression of pre-prorennin in a 
host cell is obtained by generating a DNA sequence that codes for 
pre-prorennin. That sequence has attached to it a transcriptional promoter 
and a ribosomal binding site at the 5' end and the distance between the 
beginning of the DNA that codes for pre-prorennin and the segment of DNA 
carrying the promoter and binding site is varied. The DNA is then 
transformed into host cells. The host cells are cloned and those that have 
high levels of expression of pre-prorennin are selected. 
In a method of obtaining expression of prorennin or rennin in host cells, a 
DNA sequence that codes for pre-prorennin and having a 5' end is selected. 
A portion is removed from the 5' end which portion codes for the prorennin 
or rennin precursor polypeptide. The remainder bearing the prorennin or 
rennin coding sequence is ligated onto a synthetic piece of DNA carrying a 
translational initiation codon at the 3' end of the piece. One then 
proceeds as before by attaching a transcriptional promoter and ribosomal 
binding site to the sequence and varying the distance between the 
beginning of the DNA that codes for prorennin or rennin and the segment of 
DNA carrying the promoter and ribosome binding site. This material is 
transformed into host cells, cloning is carried out and selection of the 
cells that express prorennin or rennin as desired and selected above is 
carried out. 
Escherichia coli prepared by a process described herein are exemplified by 
a culture deposited in the American Type Culture Collection of 12301 Park 
Warren Drive, Rockville, Md. 20852 and identified as Accession No. 31929 
which is strain CGE24 a derivative of E. coli strain BNN45. 
Yeast microorganisms prepared by the process described herein are 
exemplified by cultures deposited in the American Type Culture Collection 
of 12301 Park Warren Drive, Rockville, Md. 20852 and identified as 
Accession No. 20623 which is strain CGY116 a derivative of Saccharomyces 
cerevisiae strain CGY80. 
Preferably in the methods of this invention pre-prorennin, prorennin and 
rennin can each by obtained by isolation of pre-prorennin DNA material. 
The pre-prorennin is a precursor of prorennin and is not described in the 
literature. By removing portions of the pre-prorennin DNA, one could 
obtain genetic material which will code for prorennin or for rennin. 
Pre-prorennin, prorennin or rennin genes in accordance with this invention 
comprise any nucleotide sequences coding for the amino acid sequence of 
pre-prorennin, prorennin or rennin respectively and exclude any 
intervening nucleotide sequences present in the genomic DNA encoding 
pre-prorennin, prorennin or rennin respectively. These three genes are 
also provided attached to vectors which replicate in suitable host cells. 
The cells are preferably E. coli which are capable of expressing 
recombinant DNA material to produce the desired enzymatic protein. Yeast 
and other cells can also be used. These cells are selected to be capable 
of producing large quantities of the enzymatic protein under reasonable 
commercial culture conditions. 
The enzyme rennin (EC 3.4.23.4), which is referred to in this application 
is also known as chymosin. It is the major proteolytic protein found in 
the stomach of the pre-ruminant calf and is responsible for clotting milk. 
Rennin is used commercially for the production of cheese. Prorennin is a 
precursor form of rennin having 42 additional amino acids at the amino 
terminal end as described by B. Foltmann et al, Proc. Nat. Acad. Sci. USA 
74 2321-2324 (1977). Pre-prorennin, first described in this application, 
is a precursor form of prorennin and has a number of additional amino 
acids (preferably 16 amino acids) on the amino terminal end of the 
prorennin molecule. These additional amino acids are probably important 
for secretion of the enzyme from the stomach cells. B. Foltmann and others 
have shown that purified rennin is a mixture of two forms, A and B, (B. 
Foltmann et al, Proc. Nat. Acad. Sci. USA 74 2321-2324 (1977) and J. Biol. 
Chem. 254 8447-8456 (1979). Both forms are active, and sequencing data 
indicates that probably the only difference is an aspartate residue at 
position #290 in rennin A and a glycine residue at that position in rennin 
B. The rennin produced in the examples of this invention is rennin A; 
however, the same procedures and/or simple conversions can enable 
production of rennin B. Similarly the pre and pro forms may occur in an A 
or B form. 
For the purposes of this application, the prorennin gene is defined as any 
sequence of nucleotides which codes for the prorennin molecule, the amino 
acid sequence of which is described in the literature (B. Foltmann, V. B. 
Pedersen, H. Jacobsen, D. Kauffman, and G. Wybrandt, Proc. Nat. Acad. Sci. 
USA 74, 2321-2324 (1977). 
The pre-prorennin gene includes the sequence of nucleotides coding for 
prorennin, but also includes 48 additional nucleotides on the 5' end which 
code for the amino-terminal precursor polypeptide found on the 
pre-prorennin enzyme. 
The rennin gene is defined as any sequence of nucleotides which code for 
the prorennin molecule excluding the first 126 nucleotides which encode 
the proenzyme portion of prorennin. 
The living cells are prepared in the first instance by using a plurality of 
known DNA technologies starting with materials obtained from the fourth 
stomach of a calf. 
It is a feature of this invention that the rennin, pre-prorennin and 
prorennin obtained can be used in cheese-making to clot milk to obtain 
cheese and perhaps in other commercial applications to clot milk to obtain 
cheese. Large volumes can be produced by culture techniques. Thus large 
amounts of materials are capable of being produced at reasonable 
production rates and costs. The genetic recombinant DNA material is 
substantially identical to the rennin portion of the calf pre-prorennin 
gene when rennin is to be produced, substantially identical to the calf 
pre-prorennin gene when pre-prorennin is to be produced and substantially 
identical to the prorennin portion of the calf pre-prorennin gene when 
prorennin is to be produced. The differences in the recombinant DNA 
material relate mainly to the molecules being devoid of introns that may 
exist in the calf gene. 
Any species of bacteria which is considered safe for recombinant DNA work 
can be used, including, for example, Escherichia coli, various species of 
Bacillus such as Bacillus subtilis, various Lactobacillus species, and 
various Micrococcus species such as Micrococcus fragilis. Other cells such 
as fungi, yeast or mammalian cells can also be used as host cells. In each 
case, the genetic information of the cells which result, contain new 
genetic material derived from recombinant DNA material. This material is 
often contained in the form of a plasmid which is capable of replicating 
in the host cell and has inserted therein genetic material from a donor 
cell at some initial stage. Once the recombinant DNA molecule is formed 
and inserted into the host cell, that host cell grows and reproduces by 
essentially normal means. Production of the enzymatic protein which the 
recombinant DNA material encodes, which can be rennin, pre-prorennin or 
prorennin, occurs in accordance with this invention.

DESCRIPTION OF PREFERRED EMBODIMENTS 
The host cells into which the desired recombinant DNA material is 
introduced are preferably derivatives of E. coli K-12. Useful derivatives 
are as follows: 
HB101, H. W. Boyer & D. Roulland-Dussoix (1969) J. Mol. Biol. 41 459-472, 
C600, M. Mandel & A. Higa J. Mol. Biol. 53 159-162 (1970), and derivatives 
of C600 such as: 
MV1 and MV12, V. Hershfield, H. W. Boyer, C. Yanofsky, M. A. Lovett & D. R. 
Helinski (1974) Proc. Natl. Acad. Sci. USA 71 3455-3459, 
LE392, S. M. Tilghman, D. C. Tiemeier, F. Polsky, M. H. Edgell, J. G. 
Seidman, A. Leder, L. W. Enquist, B. Norman & P. Leder (1977) Proc. Natl 
Acad. Sci. USA 74 4406-4410, 
JM101, J. Messing (1979) Recombinant DNA Technical Bulletin 2 43-48, 
W3110 and derivatives, K. L. Korn & C. Yanofsky (1976) J. Mol. Biol. 103 
395-409. 
The cells can be grown by conventional culturing techniques. For example, 
culture media for the E. coli can be: 
______________________________________ 
per liter 
______________________________________ 
Rich media: 
LB 10 g Bacto tryptone (Difco Laboratories, Detroit) 
5 g Bacto yeast extract (Difco Laboratories, Detroit) 
10 g NaCl 
2 g glucose 
TY 10 g Bacto tryptone (Difco Laboratories, Detroit) 
1 g Bacto yeast (Difco Laboratories, Detroit) 
8 g NaCl 
1 g glucose 
Minimal media: 
M9 NaHPO.sub.4 6 g 
KH.sub.2 PO.sub.4 
3 g 
NaCl 0.5 g 
NH.sub.4 Cl 1 g 
CaCl.sub.2 11 mg 
MgSO.sub.4 7H.sub.2 O 
0.2 g 
Bl(thiamine 5 mg 
HCl) 
amino acids as required by the strain (40 mg each) 
glucose 2 g 
______________________________________ 
The cultures can be grown in suspension, plated on agar medium, or other 
standard tissue and cell culture techniques can be used. 
The culture media used can be any of the standard culture media for growing 
the particular cells. For example, TY medium with the cells initially 
seeded at a level of 1% to 4% where E. coli LE392 (E. coli C600 
r.sub.k.sup.- m.sub.k.sup.+ SupE, SupF, gal.sup.-) or BNN45 (E. coli 
hsdR.sup.-, hsdM.sup.+, SupE, SupF, Bl.sup.- met.sup.-) (Advanced 
Bacterial Genetics, R. W. Davis, D. Botstein, J. R. Roth, Cold Spring 
Harbor Laboratory [1980] p. 7) is preferred. 
In standard techniques, the E. coli are grown to a density of from 10 to 
30.times.10.sup.12 cells/liter and the desired enzymatic protein is 
produced. 
Various media known for growing cells can be used and form no part of the 
present invention. Similarly a variety of growing methods, techniques and 
conditions can be used. For example, while the cells are preferably grown 
at temperatures of from 30 degrees C. to 40 degrees C., temperature 
outside of this range can be used. 
The starting point for obtaining the cells of the present invention is the 
use of recombinant DNA techniques known in the art to obtain the genetic 
material desired and to insert it into the host cell after which the host 
cell is cloned. 
Preferably, the rennin gene, pre-prorennin gene or prorennin gene which one 
wishes to ultimately clone in an organism is isolated in a first step by 
obtaining messenger RNA of the pre-prorennin gene from a tissue source. In 
the case of the calf, this is obtained by isolation from the fourth calf 
stomach. The messenger RNA can be isolated as by the method of Deeley et 
al (R. G. Deeley, J. I. Gordon, A. T. H. Burns, K. P. Mullinix, M. 
Bina-Stein, R. F. Goldberger J. Biol. Chem. 252 8310-8319 [1977]) and poly 
A-enriched RNA can be obtained by chromatography over oligo (dT) cellulose 
by the method of R. C. Desrosiers, K. H. Friderici, & F. M. Rottman 
Biochemistry 14 4367-4374 (1975). 
The messenger RNA is then converted to double-stranded DNA by conventional 
means. First, the complimentary copy of the DNA is made from the messenger 
RNA by conventional recombinant DNA means as by the use of AMV reverse 
transcriptase. For example, the methods of A. Efstratiadis, F. C. Kafatos, 
A. M. Maxam and T. Maniatis, Cell 7 279-288 (1976), R. Higuchi, G. V. 
Paddock, R. Wall and W. Salser, Proc. Nat. Acad. Sci. USA 73, 3146-3150 
(1976), D. L. Kacian and J. C. Myers, Proc. Nat. Acad. Sci. USA 73, 
2191-2195 (1976), M. P. Wickens, G. N. Buell and R. T. Schimke, J. Biol. 
Chem. 253, 2483-2495 (1978), G. M. Wahl, R. A. Padgett and G. R. Stack, J. 
Biol. Chem., 254, 8679-8689 (1979) can be used to obtain the copy DNA 
(cDNA). The RNA portion can be disposed of by breaking the strands as 
known in the art using any of the above methods or by heat denaturing 
according to the method of Wickens et al (1978 ). 
Next, enzymes such as E. coli DNA polymerase I or AMV reverse transcriptase 
can be used to turn the cDNA into double-stranded DNA using the methods of 
the publications above and J. I. Gordon, A. T. H. Burns, J. L. Christmann 
& R. G. Deeley, J. Biol. Chem. 253, 8629-8639 (1978). 
Thirdly, synthetic linkers can be attached to both ends of the 
double-stranded DNA as for example by the use of Hind III or Eco R1 
synthetic oligonucleotide linkers using conventional methods such as 
described in R. H. Scheller, T. L. Thomas, A. S. Lee, W. H. Klein, W. D. 
Niles, R. J. Britten and E. H. Davidson, Science 196, 197-200 (1977), T. 
H. Fraser and B. J. Bruce, Proc. Natl. Acad. Sci. USA 75 5936-5940 (1978), 
A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter & 
H. M. Goodman, Science 196, 1313-1319 (1977), J. Shine, P. H. Seeburg, J. 
A. Martial, J. D. Baxter & H. M. Goodman, Nature 270, 494-499 (1977), or 
P. H. Seeburg, J. Shine, J. A. Martial, J. D. Baxter & H. M. Goodman, 
Nature 270, 486-494 (1977). 
In a fourth step, the DNA molecule is integrated into the chromosome or 
attached to a vector which can be a plasmid, virus or cosmid as known in 
the art. Such vectors include: 
pBR322 (F. Bolivar, R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. 
Heyneker, H. W. Boyer, J. H. Crosa, S. Falkow, 1977 Gene 2 95-119) 
pMB9 (R. L. Rodriguez, F. Bolivara, H. M. Goodman, H. W. Boyer, M. C. 
Betlach in "Molecular Mechanisms in the Control of Gene Expression" [D. P. 
Nierlich, W. J. Rutter, C. F. Fox, Eds.] 471 Academic Press New York 1976) 
pSC101 (S. N. Cohen, A. C. Y. Chang, H. W. Boyer, R. B. Helling 1973 Proc. 
Nat. Acad. Sci. USA 70 3240) 
.lambda.gtWES (D. Tiemeier, L. Enquist, P. Leder Nature 263 526-527) (1976) 
.lambda.charon phages (F. R. Blattner, et al Science 196 161-169) (1977) 
f1 R229 (J. D. Boeke Molec. Gen. Genetics 181, 288-291) (1981) 
pJC75-58 (J. Collins Methods in Enzymology 68 309-326) (1979) 
This step is again carried out outside of the final host cell. Useful 
techniques for this procedure are described in the reference above in 
connection with the linkers as well as in the following publications: V. 
Hershfield, H. W. Boyer, C. Yanofsky, M. A. Lovett & P. R. Helinski, Proc. 
Natl. Acad. Sci. USA 71, 3455-3459 (1974), N. E. Murray & K. Murray, 
Nature 251, 476-482 (1974), F. R. Blattner et al, Science 196, 161-169 
(1977). 
In a fifth step, the recombinant DNA molecules can be introduced into the 
cytoplasm of the host cell line using conventional procedures such as 
described in M. Mandel & A. Higa (1970) J. Mol. Biol. 53 159-162, P. C. 
Wensink, D. J. Finnegan, J. E. Donelson & D. S. Hogness, Cell 3, 315-325 
(1974), S. N. Cohen, A. C. Y. Chang and L. Hsu, Proc. Natl. Acad. Sci. USA 
69, 2110-2114 (1972), H. M. Goodman, and R. J. MacDonald, Methods in 
Enzymology 68, 75-90 (1979), E. M. Lederberg and S. N. Cohen, J. Bact. 
119, 1072-1074 (1974). 
Recognition of the correct clone may be accomplished by the method of 
hybridization selection or by probing with synthetic oligonucleotides, (T. 
Taniguchi, Y. Fujii, Kuriyama and M. Muramatsu, Proc. Natl. Acad. Sci. USA 
77, 4003-4006 (1980), R. P. Ricciardi, J. S. Miller & B. E. Roberts, Proc. 
Natl. Acad. Sci. USA 76, 4927-4931 (1979), D. L. Montgomery, B. D. Hall, 
S. Gillam and M. Smith, Cell 14, 673-680 [1978]). 
The newly modified host cell is then cloned and expression of the material 
desired obtained. For example, the technique of Guarente et al using the 
lactose operon promoter, (1980) (L. Guarente, G. Lauer, T. M. Roberts & M. 
Ptashne, Cell 20, 543-553 [1980], L. Guarente, T. M. Roberts & M. Ptashne, 
Science 209, 1428-1430 [1980]) allows one to obtain and optimize 
expression of foreign DNA. Other promoters can be used to obtain 
expression as known in the art so long as that promoter is active in the 
desired bacterial, yeast or other host cell. Such promoters include the E. 
coli tryptophan operon, or beta-lactamase promoters, and S. cerevisiae, 
uracil 3 or invertase promoters. 
In a specific example of this invention, recombinant E. coli strains can be 
obtained which produce pre-prorennin A, prorennin A or rennin A as 
follows: 
1. Isolation of the RNA 
Stomach tissue from milk-fed calves was obtained fresh from a local 
slaughterhouse; the mucosa of the fourth stomach was dissected away from 
the stomach wall and frozen in dry ice. Twenty-one grams of the mucosal 
tissue was disrupted by means of a blender into 200 ml of cold buffer (10 
degrees C.) consisting of 50 mM Tris.HCl, pH 7.5, 8M guanidine HCl and 1 
mM dithiothreitol. Insoluble material was removed by centrifugation in a 
Sorvall SA-600 rotor at 10,000 rpm for 12 minutes. To the 200 ml of 
supernatant from the spin was added 100 ml of ice cold absolute ethanol. 
After 1.5 hours at -20 degrees C., the precipitate was pelleted by a 
centrifugation at 3000 rpm for 30 minutes at -10 degrees C. The pellet was 
dissolved in 40 ml of ice cold buffer (EGAD) consisting of 20 mM EDTA, 
pH7, 20 mM NaOAc, pH7, 8M guanidine.HCl, and 1 mM dithiothreitol. Twenty 
milliliters of cold absolute ethanol was added and the solution placed at 
-20 degrees C. for 45 minutes. The precipitate was pelleted by 
centrifugation at 3000 rpm for 20 minutes at -10 degrees C. The pellet was 
redissolved in 40 ml cold EGAD buffer and the precipitation with 20 ml 
cold ethanol, centrifugation and redissolving the pellet in EGAD buffer 
was repeated two additional times. Finally, the pellet was dissolved in 16 
ml of 20 mM EDTA, pH7 and extracted three times with chloroform:isobutanol 
(4:1). Next, two volumes of 4.5M NaOAc pH5.2 was added to the aqueous 
layer and the solution was placed at -20 degrees C. overnight. The RNA 
precipitate was collected by centrifugation at 10,000 rpm for 25 minutes 
at -10 degrees C., and was dissolved in 30 ml water. The yield was 45 mg 
RNA. The RNA was precipitated by addition of 1 ml of 2M NaOAc pH5 and 75 
ml absolute ethanol, followed by incubation at -20 degrees C. overnight. 
The RNA was pelleted by centrifugation (10,000 rpm, 10 minutes -10 degrees 
C.) and redissolved in 20 ml water, heated to 60 degrees C. for 10 
minutes, chilled rapidly on ice and diluted with 21 ml of 2.times. 
concentrated binding buffer (20 mM Tris.HCl pH7.5, 2 mM EDTA pH7, 0.4% SDS 
and 0.24M NaCl). The RNA was applied to a 4 ml oligo-dT-cellulose column, 
the column was washed with 45 ml of 1.times. concentrated binding buffer, 
and then the poly A-containing RNA was eluted by washing the column with 
binding buffer containing no NaCl. About 1 mg of poly A-containing RNA was 
obtained. A portion of the poly A-containing RNA was translated in vitro 
in a rabbit reticulocyte lysate system (H. R. B. Pelham and R. J. Jackson 
[1976] Eur J. Biochem. 67 247- 256). The protein products were analyzed on 
a 10% polyacrylamide gel. A single major protein band was observed which 
was precipitated with rennin antiserum showing that rennin mRNA is present 
in the poly A-containing RNA. 
2. Preparation of Double-Stranded Copy DNA (cDNA) 
About 8.7 .mu.g of cDNA was synthesized from 20 .mu.g of the calf stomach 
poly A-containing RNA by incubation for one hour at 42 degrees C. in 50 mM 
Tris HCl pH8.3, 10 mM KCl, 8 mM MgCl.sub.2, 0.4 mM dithiothreitol, 1 mM 
each deoxynucleoside triphosphate, 20 .mu.g/ml oligo(-dT).sub.12-18 
containing 100 units reverse transcriptase and 1 Ci/mmole .alpha..sup.32 
P-dCTP. After heating the reaction mixture at 100 degrees C. for 3 
minutes, chilling on ice for 3 minutes and removing the precipitated 
protein by centrifugation, to half the supernatant material was added 
Hepes.KOH pH6.9 to 100 mM, MgCl.sub.2 to 5 mM, dithiothreitol to 0.5 mM, 
deoxynucleoside triphosphates to 0.125 mM. Incubation of this mixture with 
300 units of E. coli DNA polymerase I for 2 hours at 16.degree. C. 
produced 8.6 .mu.g of double-stranded cDNA. The DNA was phenol extracted 
and separated from unincorporated triphosphates by chromatography on 
Sephadex G-100 (12 ml column, 0.7 cm.times.30 cm, eluted with 20 mM 
Tris.HCl pH7.5, 0.5 mM EDTA) and was ethanol precipitated overnight at -20 
degrees C. by addition of 1/10 volume 2M NaOAc pH5, and 2.5 volumes cold 
ethanol. The double-stranded cDNA (4.6 .mu.g) was then treated with 1000 
units of S1 nuclease at 37 degrees C. for 1 hour in Buffer S (0.3M NaCl, 
30 mM NaOAc, pH4.6, 3 mM ZnSO.sub.4). The reaction was terminated by 
addition of EDTA to 10 mM, and Tris.HCl pH8.3 to 200 mM, and the mixture 
applied to a Biogel A-150 m column (0.7 cm.times.33 cm) equilibrated and 
eluted with 10 mM Tris.HCl pH7.5, 1 mM EDTA and 250 mM NaCl. The peak 
fractions (0.5 ml each) of large molecular weight DNA were pooled and 
ethanol precipitated by addition of 1/10 volume 2M NaOAC pH5 and 2.5 
volumes cold absolute ethanol. 
3. Addition of Hind III Linkers 
The S1-treated double-stranded cDNA (1.7 .mu.g) was incubated in Buffer T 
(25 mM Tris.HCl pH8, 6.6 mM MgCl.sub.2, 0.5 mM EDTA, 5 mM 
2-mercaptoethanol and 0.5 mM of each deoxynucleoside triphosphate) with 2 
units of T.sub.4 DNA polymerase at room temperature for 30 minutes. The 
material was phenol extracted and ether extracted and ethanol precipitated 
by addition of 1/10 volume 2M NaOAc pH5 and 2.5 volumes ethanol. This 
blunt-ended double-stranded cDNA was next incubated in 66 mM Tris.HCl 
pH7.6, 6.6 mM MgCl.sub.2, 5 mM 2-mercaptoethanol, 0.5 mM ATP, with 300 
pmoles of .sup.32 P-labelled Hind III synthetic linker (100.times.excess 
over cDNA ends) and 9 blunt-end units of T.sub.4 DNA ligase at 12 degrees 
overnight. 
The reaction was adjusted to 10 mM EDTA pH8 and fractionated on a Biogel 
A-150 m column (0.7 cm.times.20 cm). Fractions (0.25 ml each) containing 
high molecular weight DNA were pooled and ethanol precipitated. This 
material was treated with Hind III restriction endonuclease (9 units) in 
5.6 mM Tris.HCl pH7.6, 5.6 mM MgCl.sub.2 at 37 degrees C. for 45 minutes, 
then phenol extracted, ether extracted and ethanol precipitated by the 
addition of 1/10 volume 1M NaOAc pH5 and 2.5 volume, absolute ethanol. 
This double-stranded cDNA with Hind III cohesive termini was then ligated 
to f1 phage CGF4 double-stranded DNA which had been cut open with Hind III 
restriction endonuclease and treated twice with calf intestinal 
phosphatase by the method of H. Goodman and R. J. MacDonald (H. M. Goodman 
and R. J. MacDonald [1979] Methods in Enzymology 68, 75-91) to remove the 
terminal phosphates (Note: In order to produce phage OCF4, f1 phage R229 
(J. D. Boecke [1981] Mol. Gen. Genet. 181, 288-291) was cut with EcoRI 
endonuclease, rendered blunt-ended with T4 DNA polymerase and ligated with 
Hind III synthetic oligonucleotide linkers from Collaborative Research, 
Inc. of Waltham, Mass.). The ligation reaction contained 66 mM Tris.HCl 
pH7.6, 6.6 mM MgCl.sub.2, 5 mM 2-mercapto-ethanol, 0.3 .mu.g 
double-saturated cDNA, 0.2 .mu.g CGF4 DNA, 0.5 mM ATP and 300 cohesive-end 
units of T.sub.4 DNA ligase. Ligation was for 29 hours at 16 degrees C. 
4. Transfection of E. coli BNN45 with recombinant-OGF4 DNA 
E. coli strain CGE6 (BNN45; hsdR.sup.-, hsdM.sup.+, sup E, sup F, Bl.sup.-, 
met.sup.-) was grown in tryptone broth at 37 degrees C. with shaking and 
harvested at OD.sub.700 '0.5 by centrifugation at 7000 rpm for 10 minutes 
at 4 degrees C. The cells were resuspended in ice cold 50 mM CaCl.sub.2 
(one-half the original culture volume) and allowed to sit at 0 degrees C. 
for 30 minutes. The suspension was then centrifuged at 7000 rpm for 10 
minutes at 4 degrees C. and resuspended in 1/20 the original culture 
volume ice cold 50 mM CaCl.sub.2. After standing at 0 degrees C. for 60 
minutes the cells were used for transfection. One-half microliter of the 
20 .mu.l ligation reaction was added to each of 8 tubes containing 50 
.mu.l sterile 50 mM Tris.HCl pH7.6. One-tenth milliliter of the CaCl.sub.2 
-treated cells was added to each tube and the mixtures sat on ice for 30 
minutes. After warming to 37.degree. C. for two minutes, 0.2 ml of a CGE5 
(JM101: J. Messing [1979], F'tra D36 pro AB lac IZ.gradient.M15 in a 
.gradient.(lac pro) SupEthi.sup.- background) overnight culture and 3 ml 
of 0.7% soft agar were added, and the mixture poured onto eight tryptone 
agar plates. Incubation at 37 degrees C. overnight produced about 250 
plaques per plate. 
5. Identification of a Recombinant CGF4 Carrying the Rennin Coding Sequence 
The plaques were transferred to nitrocellulose and probed as described by 
Benton & Davis (W. D. Benton and R. W. Davis [1977] Science 196, 180-182) 
using .sup.32 P-labelled cDNA made from the calf-stomach poly A-containing 
RNA using .alpha..sup.32 P-dCTP and reverse transcriptase (T. P. St. John 
and R. W. Davis [1979] Cell 16 443-452). Abou 80 recombinant phage which 
hybridize intensely to the labelled cDNA were picked from the plates and 
stored in TY medium at 4 degrees C. Samples of the intact phage were 
amplified by growth overnight on CGE5 cells, harvested by centrifugation, 
and subjected to electrophoresis in a 2% agarose gel containing 0.37M 
Tris.glycine pH9.5 and stained with ethidium bromide after treatment in 
0.2N NaOH for one hour and neutralization in 0.5M Tris HCl pH7.4. The 
migration is inversely proportional to the log of the size of the phage 
DNA and allowed selection of eight phage carrying inserted DNA of size 
1000 to 2000 base pairs. Double-stranded RFI DNA was prepared from these 
eight phages by the method of Moses et al (P. B. Moses, J. D. Boeke, K. 
Horiuchi & N. D. Zinder [1980] Virology 104, 267). This DNA was cut with 
Hind III and the resulting fragments analyzed on an agarose gel to confirm 
that the insert was in the Hind III site and of the anticipated size. 
Finally, the DNA from four of the recombinant phages (approximately 5-10 
.mu.g from each) and DNA from the vector CGF4 was cut with Hind III and 
the fragments, after denaturation by boiling for 45 seconds and freezing 
in dry ice/ethanol, were bound to nitrocellulose by spotting the DNA in 
water onto small pieces of nitrocellulose pretreated with 20.times. SSC 
and dried. After baking in vacuo at 75 degrees C. for 1.5 hours, the DNA 
bound to nitrocellulose was carried through the hybrid selection procedure 
as described by Miller et al (J. S. Miller, R. P. Ricciardi, B. E. 
Roberts, B. M. Paterson & M. B. Mathews [1980] J. Mol. Biol. 142, 455-488) 
using 2 .mu.g poly A-enriched calf stomach RNA for each hybridization. The 
eluted RNA was then translated in a reticulocyte lysate system labelling 
with .sup.35 S-methionine by the method of Pelham and Jackson (H. R. B. 
Pelham & R. J. Jackson [1976] Eur. J. Biochem. 67, 247-256) and the 
resulting protein products analyzed on a 10% Polyacrylamide gel containing 
0.1% SDS according to Laemmli (U. Laemmli [1970] Nature 227, 680-685). The 
results of the gel analysis indicated that all four of the phage DNAs 
tested did hybridize to the rennin mRNA since all four selected an RNA 
species which, upon translation in a rabbit reticulocyte-lysate, yields a 
protein product identical to pre-prorennin in size and immunological 
criteria. Two of the four, 293-207 which has an insert of about 1400 base 
pairs (bp) and 293-118/37 which has an insert of about 1250 bp, were 
chosen for further study. The DNA inserts were sequenced by the method of 
Maxam and Gilbert (A. M. Maxam and W. Gilbert [1980] Methods of Enzymology 
68, 499-560). From nucleotide 205 to 1350 is the DNA sequence for the 
pre-prorennin A gene (see Table 1). The nucleotide sequences 1-204 and 
1351 to 1460 are attached to the pre-prorennin but can be removed if 
desired and are not essential to use of the gene in expression. Useful 
portions of the DNA material of Table 1 can be separated and used by known 
techniques. 
3 TABLE 1 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
##STR15## 
##STR16## 
##STR17## 
This Table combines information from both 293-207 and 293-118/37: 
recombinant phage 293-207 carries an insert bearing the sequence shown in 
Table 1 from nucleotide #1 to at least nucleotide #1360 except for 
nucleotides 848-961 which are deleted, while phage 293-118/37 carries an 
insert bearing the sequence from nucleotide #229 to nucleotide #1460. As 
revealed by the sequencing results, initiation of rennin synthesis occurs 
at a methionine codon (nucleotides 205-207) and results in a pre-prorennin 
molecule with sixteen additional amino acids compared to purified 
prorennin (The prorennin B amino acid sequence was published by B. 
Foltmann et al. Proc. Nat. Acad. Sci. USA 74 2321-2324 (1977) and B. 
Foltmann et al J. Biol. Chem. 254 8447-8456 (1979); the nucleotide 
sequencing data of Table 1 is the first indication for the existence of 
pre-prorennin). Together, the two recombinant f1 phages 293-207 and 
293-118/37 carry the DNA sequence for the entire pre-prorennin A molecule. 
The prorennin portion of the pre-prorennin A differs from prorennin B at 
amino acid #290 (aspartate in rennin A and glycine in rennin B as 
described by Foltmann et al [see above]; amino acid position numbering is 
that of Foltmann). An asparagine codon is shown at amino acid position 
#204 while Foltmann reported an aspartate at that position; however, this 
may be an amino acid sequencing error since the amides of aspartate and 
glutamate are difficult to distinguish from their acid forms, while 
nucleotide sequencing can readily distinguish the codons. 
The cloned rennin gene represented by phage 293-118/37 was used to 
investigate properties of the bovine genomic copy or copies of the rennin 
gene. These experiments were done by hybridizing cloned rennin DNA 
labelled with .sup.32 P by the method of nick-translation (P. W. J. Rigby, 
M. Dieckmann, C. Rhodes, and P. Berg [1977] J. Mol. Biol. 113, 237-251) to 
bovine DNA cut with various restriction enzymes, separated with an agarose 
gel and transferred to a nitrocellulose membrane according to the method 
of Southern (E. M. Southern [1975] J. Mol. Biol. 98, 503-571). The results 
indicate that restriction endonuclease cleavage of the bovine DNA with 
enzymes such as SacI and BglI, which do not cut the cloned pre-prorennin 
cDNA sequence, nevertheless frequently yields more than one band of DNA 
which will hybridize to the rennin sequence. This suggests (a) that the 
genomic copy of rennin information contains additional DNA, presumably 
intervening sequences, which contain restriction enzyme sites not found in 
rennin cDNA, or (b) that more than one rennin gene exists in the genome 
and some restriction enzymes cut between the copies. This latter 
possibility was eliminated by hybridizing restriction cut bovine genomic 
DNA with .sup.32 P-labelled probes derived from the 5' and 3' ends of the 
cloned rennin cDNA. These results, using restriction endonucleases EcoRI 
and BamHI for example, are consistent with a single genomic copy of rennin 
coding information. This means that A and B forms of rennin observed by B. 
Foltmann et al (J. Biol. Chem. 254, 8447-8456 [1979]) are most likely the 
products of two different alleles of the rennin gene. Furthermore, the 
bovine genomic copy of the rennin gene contains intervening sequences, and 
in that respect the genomic copy is different from our cloned cDNA gene 
which is identical to the messenger RNA for pre-prorennin. 
6. Expression of Pre-prorennin in E. coli 
A plasmid, pCGE5, designed to facilitate obtaining expression of 
pre-prorennin in E. coli was constructed by ligation of three agarose 
gel-purified segments of DNA. The plasmid pBR322 (4.5 .mu.g) was cut with 
restriction endonucleases Hind III (N.E. Biolabs, 6 units) and Pst I (N.E. 
Biolabs, 3 units) for one hour at 37.degree. C. in a 50 .mu.l reaction 
containing 50 mM NaCl, 7 mM Tris.HCl pH 7.5, 7 mM MgCl.sub.2 and 6 mM 
2-mercaptoethanol. Double-stranded RFI DNA from recombinant phage 293-207 
(4 .mu.g) was cut with the restriction endonuclease Kpn I (N.E. Biolabs, 
10 units) for one hour at 37.degree. C. in a 50 .mu.l reaction containing 
Buffer K (6 mM NaCl, 6 mM Tris.HCl pH 7.5, 6 mM MgCl.sub.2, and 6 mM 
2-mercaptoethanol). About 4.5 .mu.g of DNA from plasmid pLG400 (L. 
Guarente, G. Lauer, T. Roberts, M. Ptasne, Cell 20, 543-553 1980) was cut 
with Hind III (N.E. Biolabs, 6 units) for one hour at 37.degree. C. in a 
50 .mu.l reaction containing Buffer H (60 mM NaCl, 7 mM Tris.HCl pH 7.5, 7 
mM MgCl.sub.2). After phenol extraction, ether extraction, and ethanol 
precipitation, the cut DNA from 293-207 and pLG400 were separately treated 
with T.sub.4 DNA polymerase (P-L Biochemicals, 10 units) for 30 minutes at 
37.degree. C. in a 50 .mu.l reaction containing Buffer T (25 mM Tris.HCl 
pH 8, 6.6 mM MgCl.sub.2, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, and 0.5 mM 
of each deoxynucleotide triphosphate) in order to create blunt ends at the 
restrction cuts. After phenol extraction, ether extraction and ethanol 
precipitation, the pLG400 DNA was further cut with Pst I (N.E. Biolabs, 3 
units) for two hours at 37.degree. C. in 50 .mu.l of Buffer P (50 mM NaCl, 
7 mM Tris.HCl pH 7.5, 7 mM MgCl.sub.2, and 6 mM 2-mercaptoethanol) while 
the 293-207 DNA was cut with Hind III (N.E. Biolabs, 6 units) for two 
hours at 37.degree. C. in 50 .mu.l of Buffer H. Each of the three 
preparations of restriction cut DNA was phenol extracted, ether extracted, 
and ethanol precipitated, and redissolved in 30 .mu.l H.sub.2 O and 
applied to a preparative horizontal 1% agarose gel. After electrophoresis 
for 3-4 hours at 70-80 volts in 40 mM Tris.acetate pH 7.2, the gel was 
stained with ethidium bromide and examined under long wavelength 
ultraviolet light. The 500 base pair (bp) band from 293-207, the 6000 bp 
band from pLG400, and the 800 bp band from pBR322 was excised and the DNA 
extracted by freezing and thawing the gel pieces (Thuring et al, Anal. 
Biochem. 66, 213 [1975]). All three DNA segments were ethanol precipitated 
and redissolved in H.sub.2 O. Approximately 0.15 pmoles of each piece was 
ligated together overnight at 14.degree. C. in a 20 .mu.l reaction 
containing Buffer L (66 mM Tris.HCl pH 7.5, 6.7 mM MgCl.sub.2, 10 mM 
dithiothreitol, 0.75 mM ATP) and T.sub.4 DNA ligase (N.E. Biolabs, 600 
units). Transformation-competent E. coli strain CGE6 cells were prepared 
exactly as described in Section 4, and 5 .mu.l of the ligated DNA in 50 
.mu.l of 50 mM Tris.HCl pH 7.6 was mixed with 100 .mu.l of the cells for 
one hour at 0.degree. C., heat treated at 37.degree. C. for two minutes, 
and diluted ten-fold with fresh tryptone broth. After incubation for one 
hour at 37.degree. C. with shaking, cells were plated on tryptone plates 
containing ampicillin (20 .mu.g/ml). Ampicillin-resistant colonies were 
picked, and the plasmid DNA was prepared and analyzed by restriction 
enzyme digestion. By these criteria several strains carried the desired 
plasmid, pCGE5 FIG. 1. 
DNA sequence analysis revealed that the junction between 293-207 DNA and 
pLG400 DNA was as expected, and thus, the 5' end of the pre-prorennin is 
fused in frame to the 3' end of the I"Z fusion of the Guarente et al. 
Plasmid DNA was prepared from a strain carrying pCGE5 by standard methods 
(D. B. Clewell and D. R. Helinski, Proc. Nat. Acad. Sci. USA 62 1159-1166 
[1969]). 
A DNA fragment carrying the lactose operon promoter and ribosome binding 
site was isolated from plasmid pGL101 (L. Guarente, G. Lauer, T. Roberts 
and M. Ptashne, Cell 20 543-553 [1981]) by cutting 10 .mu.g of the DNA 
with Pvu II (N.E. Biolabs, 7.5 units) and Pst I (N.E. Biolabs, 4 units) 
for two hours at 37.degree. C. in 100 .mu.l reaction containing Buffer P. 
The 850 bp segment was isolated from a preparative agarose gel by excision 
of the band and freeze/thaw as described above. 
Plasmid pCGE5 DNA (40 .mu.g) was cut with Hind III (CRI, 70 units) for one 
hour, at 37.degree. C. in a 150 .mu.l reaction containing Buffer H (see 
above). This DNA was next digested with the exonuclease Bal 31 (N.E. 
Biolabs, 5 units) for 10 minutes at 30.degree. C. in a 200 .mu.l reaction 
contaning Buffer B (0.6M NaCl, 12 mM CaCl.sub.2, 12 mM MgCl.sub.2, 20 mM 
Tris.HCl pH 8, and 1 mM EDTA) (see FIG. 2). 
Analysis by gel electrophoresis indicated that the Bal 31 treatment removed 
a sufficient number of nucleotides to yield a set of fragments having 5' 
ends near the ATG initiation codon for pre-prorennin. The DNA was rendered 
blunt ended as described above using T.sub.4 DNA polymerase, and then the 
DNA (1 82 g) was partially digested with pst I (N.E. Biolabs, 0.06 units) 
for 5 minutes at 37.degree. C. in a 20 .mu.l reaction containing Buffer P. 
Next, this DNA was ligated together with the 850 bp DNA fragment carrying 
the lactose operon promoter and ribosome binding site (0.2 .mu.g) in a 20 
.mu.l reaction containing T.sub.4 DNA ligase (CRI, 300 units) in buffer L. 
Transformation-competent cells of E. coli strain CGE7 (NK5031, 
suIII.sup.+, lac.gradient.M5265, nal.sup.4, F.sup.-, Bl.sup.-) were 
prepared exactly as described above for strain CGE6, and 100 .mu.l of the 
cells in suspension were transformed with 5 .mu.l of the reaction mix, 
incubated, heat shocked and grown for phenotypic expression of ampicillin 
resistance exactly as described above for CGE 6 transformation with pCGE5. 
The cells were plated on MacConkey lactose plus ampicillin (20 .mu.g/ml) 
medium. Dark red colonies, expressing .beta.-galactosidase were picked and 
assayed for .beta.-galactosidase activity (J. Miller, Experiments in 
Molecular Genetics New York, Cold Spring Harbor Laboratory 1972). The 
plasmid DNA was isolated from twelve of these transformants, and analyzed 
by restriction enzyme digestion and agarose or polyacrylamide gel 
electrophoresis. One strain, CGE20, bears the lactose operon promoter 
about 40 nucleotides from the ATG initiation codon of the 
pre-prorennin-I"Z fusion on plasmid pCGE17 (see FIG. 3) and produces 
intermediate levels of .beta.-galactosidase (about 1/3 of the fully 
induced level of a lactose.sup.+ strain such as CGE6). 
In order to create a plasmid bearing the entire pre-prorennin gene fused to 
the lactose promoter and ribosome binding site, pCGE17 DNA (4 .mu.g) was 
cut with Bgl II (N.E. Biolabs, 6 units) for one hour at 37.degree. C. in a 
90 .mu.l reaction containing Buffer P. Then, Tris.HCl pH 7.5 was added to 
100 mM and the DNA was further cut with EcoRI (Boehringer/Mannheim, 40 
units) for an additional hour at 37.degree. C. Next, pBR322 DNA (5 .mu.g) 
was cut with Pvu II (N.E. Biolabs, 5 units) for one hour at 37.degree. C. 
in a 45 .mu.l reaction containing Buffer P, followed by addition of 
Tris.HCl pH 7.5 to 100 mM and addition of EcoRI (Boehringer/Mannheim, 40 
units) with further incubation at 37.degree. C. for one hour. Finally, 
recombinant f1 phage 392-118/37 RFI DNA (6 .mu.g) was cut with Hind III 
(N.E. Biolabs, 6 units) for one hour at 37.degree. C. in a 50 .mu.l 
reaction containing Buffer H. After phenol extraction and ethanol 
precipitation, the cut DNA was treated with T.sub.4 DNA polymerase (P-L 
Biochemicals, 10 units) at room temperature for 30 minutes in a 50 .mu.l 
reaction containing Buffer T to blunt the Hind III site. Then, the 
redissolved phenol-extracted and ethanol precipitated DNA was cut with Bgl 
II (N.E. Biolabs, 4 units) for one hour at 37.degree. C. in a 30 .mu.l 
reaction containing Buffer P. The three restriction cut DNA species were 
applied to a preparative horizontal agarose gel, and the 370 bp pCGE17 
piece, the 2300 bp pBR322 piece and the 1000 bp 293-118/37 piece were 
excised and eluted by freezing and thawing the agarose chunk. After 
ethanol precipitation, the DNA was redissolved in water and about 0.2 
pmoles of each piece were ligated together for six hours at 14.degree. C. 
in a 20 .mu.l reaction containing Buffer L and T.sub.4 DNA ligase (N.E. 
Biolabs, 300 units). E. coli strain CGE6 was transformed with the ligated 
DNA as described above and ampicillin resistant colonies were picked. 
Analysis of the plasmid DNA by restriction enzyme cleavage revealed strain 
CGE24 carries the plasmid pCGE21 (see FIG. 4) which gears the entire 
pre-prorennin sequence fused to the lactose operon promoter and ribosome 
binding site. 
Two kinds of analysis reveal that this strain is synthesizing authentic 
calf pre-prorennin. First, crude extracts of the cells inhibit binding of 
iodinated rennin to anti-rennin serum in a radioimmune assay performed 
according to the method of Peak et al (G. J. Peak, J. Morris and M. J. 
Buckman [1979] "Growth Hormones" In Methods of Hormone Radioimmunoassay 
pp. 223-244 [B. M. Jaffe & H. R. Behrman, eds.] Academic Press, New York) 
with the following modifications: iodinated rennin is stored in 50 mM 
Sodium Phosphate pH 6., 0.15M NaCl, 1% BSA; RIA buffer is 0.05M Tris.HCl 
pH 8, 0.5% human serum albumin and 0.1% sodium nitrite; incubation is at 
4.degree. C. for 18 hours. The amount of inhibition indicates about 0.8 
.mu.g of pre-prorennin is present in each milliliter of extract (or about 
4 .mu.g of pre-prorennin per liter of cell culture). Second, cells were 
pulse labelled for 30 minutes in mid-exponential phase with .sup.35 
S-methionine, lysed and immunoprecipitated with anti-rennin serum (as 
described by J. S. Emtage et al Nature 283 171-175 [1980]). When the 
immunoprecipitates were analyzed on a 10% polyacrylamide gel containing 
SDS (U. K. Laemmli & M. Favre, J. Mol Biol. 80 575-599 [1973]) and 
autoradiographed, a band approximately the size of pre-prorennin was 
observed. In addition, a band the size of rennin was also observed, 
suggesting the bacteria may be processing the pre-prorennin to rennin, or 
a second initiation of translation may occur within the pre-prorennin 
sequence. Neither band was present in immunoprecipitates of the parent 
strain CGE6 which contains no plasmid. These results show that 
pre-prorennin is produced in E. coli cells carrying the plasmid pCGE21, 
and the level of production is about 600 molecules per cell. Higher levels 
of production will be possible using this same scheme by obtaining fusions 
of the lactose operon promoter closer to the initiation codon of 
pre-prorennin. 
Extracts of strain CGE24 were also tested for activity in the standard 
milk-clotting assay of B. Foltmann (Methods in Enzymology [1970] 19, 
421-436). The results indicate that this E. coli strain produces about 100 
molecules per cell of active rennin or an active fragment of rennin 
capable of clotting milk, while an extract from strain CGE6 which contains 
no rennin DNA sequences is incapable of clotting milk. Strain CGE24 
bearing plasmid pCGE21 is on deposit with the American Type Culture 
Collection (ATCC) Accession No. 31929. 
Strain CGE24 isE. coli strain BNN45 (hsdR.sup.- hsdM.sup.+ supE44 supF 
Bl.sup.- met.sup.-) (Advanced Bacterial Genetics, R. W. Davis, D. 
Botstein, J. R. Roth, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y. 1980 p. 7) carrying the plasmid pCGE21 which is defined by the 
diagram of FIG. 5. The plasmid contains a portion of the plasmid pBR322 
(nucleotides 2067 to 4360, see J. G. Sutcliffe [1979] Cold Spring Harbor 
Symposium 43, 77-90) and a 95 base pair fragment bounded by EcoRI and 
PvuII sites from plasmid pGL101 (L. Guarente, G. Lauer, T. M. Roberts, and 
M. Ptashne [1980] Cell 20, 543-553) fused to about 1300 nucleotides which 
code for the pre-prorennin molecule (from recombinant f1 phage 293-207 and 
293-118/37). The orientation is such that the lactose operon promoter 
drives expression of the pre-prorennin protein in E. coli. 
7. Expression of Methionine Prorennin in E. coli 
The pre-prorennin gene contains three recognition sites for the restriction 
endonuclease Hha I (recognizes GCGC [see Table 1]), one of which removes 
the "pre" signal sequence and leaves the sequence for prorennin minus the 
first nucleotide (G) for the alanine codon. Accordingly, we isolated this 
partial HhaI digestion product which represents a nearly intact prorennin 
gene. Eighteen .mu.g of RFI double-stranded DNA from recombinant phage 
293.sub.]118/37 was cut with 12 units of restriction endonuclease Hind III 
(N.E. Biolabs) in 50 .mu.l of Buffer H for one hour at 37.degree. C. The 
approximately 1230 bp insert bearing rennin DNA was purified by extracting 
the DNA from the appropriate band on a 1% agarose gel by the freeze/thaw 
method. About 1.5 .mu.g of this DNA was subjected to partial HhaI cleavage 
by incubation at 37.degree. C. for 5 minutes with 0.25 units of HhaI (N.E. 
Biolabs) in 30 .mu.l of Buffer P. DNA which corresponds to the uncut plus 
the singly cut piece missing about 25 nucleotides from the beginning of 
293-118/37 was isolated from a band on a 2% agarose gel. Plasmid pBR322 
DNA (10 .mu.g) was cut with restriction endonuclease Hind III (N.E. 
Biolabs, 9 units) in 100 .mu.l of Buffer H for one hour at 37.degree. C. 
The DNA was phenol extracted and ethanol precipitated. About 0.5 pmoles of 
each DNA (i.e., the partial HhaI cut 293-118/37 and the Hind III cut 
pBR322) were combined, redissolved in 28 .mu.l of water, and rendered 
blunt ended by treatment with DNA polymerase I (Boehringer/Mannheim, 9 
units) in a 40 .mu.l reaction containing Buffer D (60 mM Tris.HCl pH 7.5, 
8 mM MgCl.sub.2, 10 mM dithiothreitol, 1 mM ATP and 0.2 mM of each 
deoxynucleotide triphosphate) for ten minutes at 10.degree. C. A synthetic 
oligonucleotide bearing an Xba I restriction endonuclease sequence plus an 
ATGG (i.e., CCATCTAGATGG) was synthesized by the triester method (K. 
Itakura et al J. Biol. Chem. 250 4592 [1975]) by Collaborative Research, 
Inc. and 5 .mu.g was kinased with .gamma..sup.32 -P-ATP using 6 units of 
T.sub.4 polynucleotide kinase (P-L Biochemicals) in a 25 .mu.l reaction 
containing Buffer Y (70 mM Tris.HCl pH 7.6, 10 mM MgCl.sub.2, 10 mM 
2-mercaptoethanol and 2 nmoles ATP). This 5'-labelled oligonucleotide was 
added to the 40 .mu.l blunt-end reaction along with additional buffer 
components to keep the concentration constant plus 600 units of T.sub.4 
DNA ligase (N.E. Biolabs). The reaction was inducated at 14.degree. C. 
overnight, and then diluted with five volumes of a solution of 180 mM 
NaCl, 7 mM MgCl.sub.2 and 5 mM Tris.HCl pH 8. After heating at 65.degree. 
C. for five minutes, the DNA was treated with 45 units of Xba I 
restriction endonuclease (15 units added each hour for a total of three 
hours of digestion). Finally, the oligonucleotide monomers were separated 
from the large DNA by gel filtration over a Biogel A-5 m column 
(0.68.times.36 cm, see above). The excluded DNA was pooled, ethanol 
precipitated, redissolved in 12 .mu.l of water and incubated in a ligation 
reaction containing Buffer L plus 300 units T.sub.4 DNA ligase (N.E. 
Biolabs) at 14.degree. C. overnight. Five microliters of this ligation 
reaction was used to transform competent cells of strain CGE6 as described 
above. The transformed cells were plated on tryptone plates containing 20 
.mu.g/ml ampicillin, and ampicillin-resistant colonies were picked and 
screened for tetracycline sensitivity. Analysis of the plasmid DNA by 
restriction enzyme digestion (Xba I plus Kpn I) and polyacrylamide gel 
electrophoresis revealed one strain carrying the desired plasmid pCGE 181 
(i.e., gives a 250 bp Xba I-Kpn I fragment). 
About 5 .mu.g of the pCGE 181 DNA will be cut with Xba I (N.E. Biolabs, 4 
units) for one hour at 37.degree. C. in a 50 .mu.l reaction containing 
Buffer X (150 mM NaCl, 6 mM Tris.HCl pH 7.9, 6 mM MgCl.sub.2). After 
phenol extraction and ethanol precipitation, the DNA is to be rendered 
blunt ended by treatment with T.sub.4 DNA polymerase (P-L Biochemicals, 10 
unit) for 30 minutes at 37.degree. C. in a 50 .mu.l reaction containing 
Buffer T. Again, the DNA will be phenol extracted, and ethanol 
precipitated. Vector DNA is prepared by cutting 5 .mu.g of plasmid pGL101 
DNA (L. Guarente et al Cell 20 543-553 [1980]) with Pvu II (N.E. Biolabs, 
5 units) in a 50 .mu.l reaction containing Buffer P. Then, after phenol 
extraction and ethanol precipitation, the redissolved DNA will be 
phosphatased by treatment with 0.06 units calf intestinal alkaline 
phosphatase (Boehringer/Mannheim) for 30 minutes at 37.degree. C. in a 50 
.mu.l reaction containing Buffer C. The Pvu II-cut vector (0.2 pmoles) and 
the Xba I-cut prorennin DNA piece (0.2 pmoles) will be ligated together 
overnight at 14.degree. C. in a 20 .mu.l reaction containing Buffer L. 
Transformation-competent cells of strain CGE6 are prepared as described 
above and will be transformed with 5 .mu.l of the ligation reaction. The 
resulting cells are plated on tryptone agar plates containing 20 .mu.g/ml 
ampicillin. Ampicillin-resistant colonies will be picked, and the plasmid 
DNA isolated and analyzed by restriction enzyme digestion and agarose gel 
electrophoresis. A strain will be found which bears the prorennin DNA 
ligated to the lactose operon promoter and ribosome binding site such that 
prorennin protein will be made in vivo (i.e., the ATG initiation codon 
added to the prorennin sequence is nine nucleotides from the lactose 
operon robosome binding site). We will determine the amount of prorennin 
synthesized by subjecting a lysate of cells carrying the plasmid to 
radioimmunoassay using iodinated authentic purified rennin and anti-rennin 
serum. The size of the prorennin product will be determined by 
electrophoresis of immunoprecipitates of .sup.35 S-methionine labelled 
cell extracts on SDS-containing polyacrylamide gels. 
8a. Expression of Methionine-Valine-Rennin in E. coli 
In order to obtain DNA carrying only the rennin coding sequence, the RFI 
DNA from recombinant phage 293-207 was resected with the nuclease Bal 31 
and ligated into an f1 phage vector. The ligation products were cloned, 
and a library of resected rennin DNA was prepared. Specifically, 8 .mu.g 
of 293-207 phage RFI DNA was cut with 6 units Hind III (N.E. Biolabs) in a 
20 .mu.l reaction containing Buffer H for one hour at 37.degree. C. After 
phenol and ether extraction and ethanol precipitation, the DNA was 
redissolved in 20 .mu.l of water and treated with 1.25 units Bal 31 (N.E. 
Biolabs) in a 50 .mu.l reaction containing Buffer B for 30 minutes at 
30.degree. C. The reaction was stopped by phenol extraction and ethanol 
precipitation. Bal 31 resected DNA fragments in the size range 500-1000 bp 
were isolated from a 1.5% agarose gel by the freeze/thaw technique 
referred to above. The resected DNA was rendered blunt ended by treatment 
with DNA polymerase I (Boehringer/Mannheim, 9 units) in a 40 .mu.l 
reaction containing Buffer D for 10 minutes at 10.degree. C. Synthetic 
oligonucleotide linkers (specifically, Hind III 8-mer CAAGCTTG; 5.0 .mu.g 
from Collaborative Research, Inc.) were kinased with .sup.32 P-ATP using 6 
units of T.sub.4 polynucleotide kinase (P-L Biochemicals) in a 25 .mu.l 
reaction containing Buffer Y. This labelled oligonucleotide was added to 
the 40 .mu.l blunt-end reaction along with additional buffer components to 
keep the concentration constant plus 600 units T.sub.4 DNA ligase (N.E. 
Biolabs). The reaction was incubated at 14.degree. C. overnight. Next, the 
reaction was diluted five-fold with 250 .mu.l of a 60 mM NaCl plus 7 mM 
MgCl.sub.2 solution and heated at 65.degree. C. for 5 minutes. After 
cooling of the reaction mix, a total of 45 units of Hind III restriction 
endonuclease (N.E. Biolabs) was added, 15 units each hour for a total of 
three additions and three hours incubation at 37.degree. C. The 
oligonucleotide linker monomers were removed from the mixture by elution 
over a Biogel A-5 m column (0.68.times.36 cm, Bio Rad) in column buffer 
(10 mM Tris.HCl pH 7.5, 100 mM NaCl, 1 mM EDTA). The excluded peak was 
ethanol precipitated, and the DNA (about 0.5 pmoles) was added to a 20 
.mu.l ligation reaction containing Buffer L, 600 units T.sub.4 DNA ligase 
(N.E. Biolabs) and about 0.5 pmoles phage CGF4 DNA (Collaborative 
Genetics Inc.) which had been cut with Hind III and phosphatased as 
described above. After ligation at 14.degree. C. for 18 hours, 4 .mu.l of 
a 40-fold dilution of the reaction mix in 50 mM Tris.HCl pH 7.6 was used 
to transfect competent cells of strain CGE6. The transfection and plating 
for plaques was carried out exactly as described in Section 4 above. About 
500 plaques were obtained per plate; probing by the method of Benton and 
Davis (Science 196 180-182 [1977]) using nick-translated pre-prorennin DNA 
carried on plasmid pBR322 (method of P. W. J. Rigby et al [1977] J. Mol. 
Biol. 113 237-251) revealed about 15% of the plaques carried rennin DNA. 
About 250 of these were picked and stored in tryptone broth at 4.degree. 
C. Analysis of the DNA from several of these recombinant phage by 
restriction enzyme digestion and agarose gel electrophoresis will reveal 
several phage bearing Bal 31-resected pre-prorennin DNA such that the 5' 
end of the inserted sequence is close to the beginning of the rennin 
coding sequence (i.e., nucleotide 379 in the sequence given in Table 1). 
Single-stranded recombinant f1 phage DNA is isolated from these phage as 
follows. First, a plate stock of phage is prepared by infecting 0.4 ml of 
an overnight culture of CGE5 with 50 .mu.l of phage picked from a plaque. 
This is poured onto a 150 mm tryptone gear plate in 7 ml of 0.7% soft 
agar. The phage are eluted after overnight growth by adding 12 ml tryptone 
broth to the plate and incubating 2 hours. Three ml of that broth is then 
precipitated with 0.6 ml of a polyethylene glycol/NaCl solution (25% PEG 
and 2.5M NaCl) and stored at 4.degree. C. for one hour (K. R. Yamamoto et 
al Virology 40 734-744 [1970]). After centrifugation, the phage are 
resuspended in 0.3 ml Buffer TEN (10 mM Tris.HCl pH 8, 10 mM NaCl, 0.5 mM 
EDTA). Then the phage are precipitated with 30 .mu.l of the PEG/NaCl 
solution, incubated for one hour at 4.degree. C. and centrifuged. The 
phage are resuspended in 50 .mu.l Buffer TEN and 5.5 .mu.l of 1% SDS is 
added to each tube. After 55.degree. C. incubation for 10 minutes, 200 
.mu.l TEN is added and the solutions are phenol extracted, ether extracted 
and ethanol precipitated. The sequence of the inserted DNA in the 
recombinant f1 phage may be determined by the method of Sanger (F. Sanger 
et al J. Mol. Biol. 143 161-178 [1980]) using a synthetic oligonucleotide 
primer (a "universal primer" with the sequence TTGACGGGGAAAG, 
Collaborative Research, Inc., Waltham, Mass.). From the collection of Bal 
31-resected inserts cloned in f1, at least one phage will be found, to be 
called (293-207-101), which bears the Hind III linker fused to the 5' end 
of rennin at nucleotide 379 (i.e., at the codon for glycine which is the 
N-terminal amino acid of mature rennin). The RFI DNA (5 .mu.g) of the 
phage 293-207-101 will be digested with Hind III (4 units, N.E. Biolabs) 
for one hour at 37.degree. C. in a 50 .mu.l reaction containing Buffer H. 
After phenol extraction and ethanol precipitation, the DNA will be 
redissolved in 20 .mu.l water and treated with 10 units of nuclease Sl 
(Boehringer/Mannheim) at 37.degree. C. for 10 minutes in a 50 .mu.l 
reaction containing Buffer S (see Section 2 above). This DNA is again 
phenol extracted and ethanol precipitated. Next, synthetic oligonucleotide 
linkers (with the sequence, CCATCTAGATGG, 5 .mu.g, Collaborative Research, 
Inc.) are kinased with .alpha.-.sup.32 P-ATP using 6 units of T.sub.4 
polynucleotide kinase (P-L Biochemicals) in a 25 .mu.l reaction containing 
Buffer Y. This kinased oligonucleotide will be ligated onto the Hind 
III-bound insert which had been Sl-treated (as described above) and 
purified from an agarose gel by the freeze/thaw method. The ligation 
mixture will contain about 0.8 pmoles Hind III-bounded Sl-treated rennin 
DNA, 100 pmoles kinased synthetic linker and 600 units T.sub.4 DNA ligase 
(N.E. Biolabs) in 20 .mu.l Buffer L. Incubation is at 14.degree. C. for 18 
hours. The reaction will be diluted six-fold with 100 .mu.l of a solution 
of 180 mM NaCl, 7 mM MgCl.sub.2 and 5 mM Tris/HCl pH 8. After heating at 
65.degree. C. for 5 minutes, 45 units of restriction endonuclease Xba I 
(N.E. Biolabs) will be added in three additions of 15 units each during a 
three hour incubation at 37.degree. C. Oligonucleotide monomers are to be 
separated from the large DNA by gel filtration over a Biogel A-5 m column 
(0.68.times.36 cm) in column buffer (see above). The excluded DNA is 
ethanol precipitated and subjected to T.sub.4 DNA polymerase ((P-L 
Biochemicals, 10 units) treatment in 50 .mu.l of Buffer T to blunt the 
Xba-cut ends. After phenol extraction and ethanol precipitation the DNA 
will be subjected to Eco RI (N.E. Biolabs, 4 units) digestion for one hour 
at 37.degree. C. in a 50 .mu.l reaction containing Buffer R (100 mM 
Tris.HCl pH 7.5, 50 mM NaCl, 5 mM MgCl.sub.2). The rennin fragment (about 
350 bp) may be isolated from a 2% agarose gel. Phage 293-118/37 RFI DNA (5 
.mu.l) will be cut with Hind III (N.E. Biolabs, 4 units) for one hour at 
37.degree. C. in a 50 .mu.l reaction containing Buffer H. After phenol 
extraction and ethanol precipitation, the DNA should be blunt-ended at 
37.degree. C. for 30 minutes using T.sub.4 DNA polymerase (P-L 
Biochemicals, 10 units) in 50 .mu.l Buffer T. Next, following another 
phenol extraction and ethanol precipitation, the DNA will be cut with Eco 
RI (N.E. Biolabs, 4 units) for one hour at 37.degree. C. in a 50 .mu.l 
reaction containing Buffer R. This Eco RI to Hind III (blunt) DNA fragment 
from phage 293-118/37 (about 660 bp) will be isolated from a 2% agarose 
gel. Plasmid DNA (5 .mu.g) from plasmid pGL101 is cut with Pvu II (N.E. 
Biolabs, 4 units) for one hour at 37.degree. C. in 50 .mu.l of Buffer P, 
and then the DNA will be phenol extracted and ethanol precipitated. After 
treatment with calf intestinal alkaline phosphatase (0.1 unit, 
Boehringer/Mannheim) in 50 .mu.l of Buffer C, the DNA is again phenol 
extracted and ethanol precipitated. A ligation reaction will be carried 
out at 14.degree. C. for 18 hours using 0.2 pmoles of the linkered-rennin 
DNA (nucleotide 379-731), 0.2 pmoles of rennin DNA from phage 293-118/37 
(nucleotide 732-1456) and 0.2 pmoles of Pvu II cleaved pGL101 in 20 .mu.l 
of Buffer L using 300 units T.sub.4 DNA ligase (N.E. Biolabs). Five 
microliters of the reaction may be used to transform 100 .mu.l of 
Ca.sup.++ -treated CGE4 cells (as described above). Restriction 
endonuclease analysis of plasmid DNA from several ampicillin-resistant 
colonies picked from tryptone plates with 20 .mu.g/ml ampicillin will 
reveal one colony with a plasmid, pCGE188, which carries the rennin 
sequence in proper orientation to be expressed off the lactose operon 
promoter. 
We will determine the amount of rennin synthesized by subjecting a lysate 
of cells carrying the plasmid to radioimmune assay using iodinated 
authentic purified rennin and anti-rennin serum. The size of the rennin 
product will be determined by electrophoresis of immunoprecipitates of 
.sup.35 S-methionine labelled cell extracts on SDS-containing 
polyacrylamide gels. 
In addition, the amount of active rennin present in the E. coli cell 
extracts will be measured using a modified micro-scale version of the 
standard milk-clotting assay (B. Foltmann Methods in Enzymology 19 pp 
421-436 [1970]). 
8b. Expression of Methionine-Rennin A. in E. coli 
A plasmid containing the nucleotide sequence of the rennin A gene 
immediately preceded by the initiation codon ATG and under the control of 
the lac operon promoter may be constructed as described below. This 
construction requires the creation of three separate plasmids which will 
be used in stepwise recombination to produce the final product. 
The first plasmid to be made is one containing the initiation codon ATG 
immeditely preceding the first approximately 350 nucleotides of the rennin 
gene. Double-stranded recombinant f1 phage 293-118/37 DNA (200 .mu.g) was 
digested with the restriction endonuclease PstI (N.E. Biolabs, 20 units) 
for 150 minutes at 37.degree. C. in a 100 .mu.l reaction containing Buffer 
P. Eleven microliters of 100 mM Tris.HCl pH 7.5 and 4 .mu.l of EcoRI 
(Boehringer/Mannheim, 80 units/.mu.l) were added, and the digestion was 
continued at 37.degree. C. for 60 additional minutes. Restriction was 
terminated by addition of 1/10 volume of 200 mM EDTA and DNA restriction 
fragments were separated by agarose gel electrophoresis in a 0.6% agarose 
gel containing 40 mM Tris.acetate pH 8.3. The gel was stained with 
ethidium bromide (0.5 .mu.g/ml), and that portion containing the desired 
400 bp band was visualized under long wavelength ultraviolet light and 
excised. DNA was separated from the gel by the freeze-thaw method and 
ethanol precipitated. The DNA was redissolved in water and digested with 
the restriction endonuclease MspI (N.E. Biolabs, 30 units) for one hour at 
37.degree. C. in a 50 .mu.l reaction containing 10 mM Tris.HCl pH 7.4, 10 
mM MgCl.sub.2, 6 mM KCl and 1 mM dithiothreitol. This reaction produced a 
fragment containing the sequence 
##STR18## 
near the beginning of the rennin gene (the first codon of the rennin gene 
sequence is overscored, and represents nucleotides 379-381 in Table 1). 
After phenol extraction, ether extraction, and ethanol precipitation, this 
fragment and two small fragments produced by MspI digestion were treated 
with the Klenow fragment of E. coli DNA polymerase I (Boehringer/Mannheim, 
3 units) for 15 minutes at 37.degree. C. in a 42 .mu.l reaction containing 
0.05 mM deoxyadenosine triphosphate, 6.6 mM Tris/HCl pH 7.5, 6.6 mM NaCl, 
6.6 mM MgCl.sub.2 and 6.6 mM dithiothreitol. This reaction trimmed two 
nucleotides from the above sequence to produce 
##STR19## 
After phenol extraction and ether extraction the deoxyadenosine 
triphosphate was separated from the larger molecular weight species by the 
addition of 0.5 .mu.l of 200 mM spermine, incubation on ice for 15 
minutes, centrifugation for 10 minutes in a 4.degree. C. microcentrifuge, 
and centrifugation of the resulting pellet twice for five minutes each at 
4.degree. C. in the presence of 75% ethanol. The spermine was removed by 
the addition of 1 ml 75% ethanol, 0.3M sodium acetate, and 10 mM magnesium 
acetate to the pellet, followed by one hour incubation on ice, and 
centrifugation as just described. 
A second treatment of the DNA with the Klenow fragment of E. coli DNA 
polymerase I was conducted as described above except that 0.05 mM 
deoxyctylidine triphosphate was substituted for the 0.05 mM deoxyadenosine 
triphosphate of the previous reaction. This procedure produced a fragment 
with the sequence 
##STR20## 
near the beginning of the rennin gene. After phenol extraction, ether 
extraction, and ethanol precipitation, the DNA was redissolved in water 
and treated with Sl nuclease (Boehringer/Mannheim, 100 units) for 30 
minutes at room temperature in a 50 .mu.l reaction containing Buffer S. 
This enzyme removed the 5' single-stranded DNA from the fragment leaving 
the sequence 
##STR21## 
Thus, the beginning of the rennin sequence is at the 5' end of the DNA 
fragment. A synthetic oligonucleotide containing a ClaI restriction site 
and ending with the nucleotides ATG (i.e., CATCGATG, Collaborative 
Research, Inc., 5 .mu.g) was kinased with .gamma..sup.32 P-ATP using 
T.sub.4 polynucleotide kinase (P-L Biochemicals, 3 units) for 30 minutes 
at 37.degree. C. in a 25 .mu.l reaction containing Buffer Y. This kinased 
linker (about 200 pmoles was ligated to the treated DNA fragment (about 5 
pmoles) by incubation with T.sub.4 DNA Ligase (N.E. Biolabs, 900 units) at 
15.degree. C. overnight in Buffer L. The reaction was terminated by 
heating at 65.degree. C. for 5 minutes. Four microliters of 10x ClaI 
buffer (1x=10 mM Tris.HCl pH 8, 10 mM MgCl.sub.2), and 10 .mu.l of 
restriction endonuclease ClaI (Boehringer/Mannheim, 27 units) were added. 
The resulting mixture was incubated at 37.degree. C. for one hour. Four 
microliters were removed for analysis on a polyacrylamide gel, followed by 
the addition of 1 .mu.l of 10x ClaI buffer, 10 .mu.l of ClaI enzyme, and 3 
.mu.l water. This mixture was incubated for an additional hour. The 
treated DNA containing the desired rennin sequences was purified by 
separation in a 2% agarose gel containing 40 mM Tris.acetate buffer pH 
8.3. The DNA was visualized by long wave ultraviolet irradiation and 
removed from the gel by the freeze-thaw method described above. This 
fragment was then ready for insertion into the appropriate vector. 
Preparation of the vector DNA began with digestion of 3.3 .mu.g of pBR322 
DNA with 5.4 units of ClaI endonuclease (Boehringer/Mannheim) for one hour 
at 37.degree. C. in a 30 .mu.l reaction containing ClaI buffer. After 
phenol extraction, ether extraction, and ethanol precipitation, the vector 
DNA was treated with 0.06 units calf intestinal alkaline phosphatase 
(Boehringer/Mannheim) at 37.degree. C. for 15 minutes in Buffer C. After 
phenol extraction, ether extraction, and ethanol precipitation, 
approximately 1 pmole of vector DNA was mixed with approximately 2 pmoles 
of rennin fragment DNA as prepared above. These two DNA pieces were 
ligated together in a 29 .mu.l reaction containing Buffer L and T.sub.4 
DNA ligase (N.E. Biolabs, 450 units). Transformation-competent cells of E. 
colis strain CGEA3 (F.sup.- .gradient.(lac-pro)XIII, also known as strain 
LG90) were prepared as described in Section 4 and transformed with the 
ligated DNA. Ampicillin-resistant colonies selected on plates were picked, 
and the plasmid DNA was analyzed by restriction enzyme digestion. It will 
be necessary to sequence portions of these plasmids to insure that the 
proper construction containing the linker sequence, CATCGATG, adjacent to 
the beginning of the rennin gene sequence 
EQU GGG GAG . . . 
has been obtained. The plasmid with the desired correct sequence which will 
be called pCGE301 will then be used in conjunction with the other two 
plasmids described below to produce a final plasmid which will direct the 
expression of methionine-rennin in E. coli. 
Generating the second of the three plasmids required for this construction 
required the subcloning of the rennin-containing Hind III fragment of 
recombinant phage 293-118/37 double-stranded DNA. Three micrograms of 
double-stranded f1 phage 293-118/37 DNA were digested with restriction 
endonuclease Hind III (N.E. Biolabs, 3 units) for one hour at 37.degree. 
C. in a 10 .mu.l reaction containing Buffer H plus 7 mM 2-mercaptoethanol. 
Four microliters of Hind II (N.E. Biolabs, 3 units) were added and the 
mixture was incubated at 37.degree. C. for an additional hour. After 
phenol extraction, ether extraction, and ethanol precipitation, 
approximately 1.5 .mu.g of this DNA was mixed with about 1 .mu.g of pBR322 
DNA (previously treated with Hind III and calf intestinal alkaline 
phosphatase as previously described). The two DNA fragments were ligated 
together overnight at 16.degree. C. in a 20 .mu.l reaction containing 
Buffer L and T.sub.4 DNA ligase (N.E. Biolabs, 600 units). Five 
microliters of this mixture was used to transform cells of E. coli strain 
CGE6, and ampicillin-resistant colonies were isolated as described above. 
Restriction enzyne cutting and agarose gel electrophoresis revealed the 
resulting plasmid, pCGE302, consists of the prorennin gene sequence from 
phage 293-118/37 inserted into the Hind III site of pBR322. 
Generation of the third component needed for construction of the 
rennin-producing plasmid required digestion of 2 .mu.g of pGL101 (L. 
Guarente, G. Lauer, T. M. Roberts and M. Ptashne [1980] are above) DNA 
with restriction endonuclease PvuII (N.E. Biolabs, 5 units) for one hour 
at 37.degree. C. in a 20 .mu.l reaction containing Buffer H plus 10 mM 
2-mercaptoethanol. After phenol extraction, ether extraction, and ethanol 
precipitation, the DNA was mixed with a kinased synthetic oligonucleotide 
(CATOGATG, Collaborative Research, Inc., about 200 pmoles) and ligated 
with T.sub.4 DNA ligase (N.E. Biolabs, 900 units) at 16.degree. C. 
overnight in a 30 .mu.l reaction containing Buffer L. The reaction was 
terminated by treatment at 65.degree. C. for 5 minutes. Five microliters 
of this mixture was used to transform CaCl.sub.2 -treated cells of E. coli 
strain CGE43. Plasmid DNA was prepared from several transformants and 
subjected to restriction enzyme digestion and agarose gel electrophoresis 
in order to identify the desired plasmid, pCGE303, which is identical to 
plasmid pGL101 except the PvuII site has been converted to a ClaI site. 
The construction of the final plasmid containing the ATG-rennin sequence 
under transcriptional control of the lac operon promoter will involve in 
vitro recombination of the three plasmids just described and is 
theoretically outlined below. Plasmid pCGE301 will be digested with 
restriction endonucleases KpnI and Hind III, and the resulting fragments 
will be treated with calf intestinal alkaline phosphatase. Plasmid pCGE302 
will be digested with restriction endonucleases KpnI, Hind III and BglII. 
The fragments generated from these two procedures will then be mixed and 
ligated. This DNA will be used to transform E. coli strain CGE43. The 
major ampicillin-resistant plasmid product will be pCGE304, containing ATG 
attached to the entire rennin coding sequence. 
The plasmid pCGE303 will then be digested with restriction endonucleases 
PstI and ClaI and treated with calf intestinal alkaline phosphatase. 
Plasmid pCGE304 will also be digested with PstI and ClaI. DNA fragments 
resulting from these two procedures will be mixed together, ligated, and 
used to transform strain CGE43. The ampicillin-resistant plasmids derived 
from the transformed cells will be analyzed by size, restriction enzyme 
digestion, and DNA sequence to find the desired plasmid pCGE305 which will 
bear the ATG-rennin sequence under transcriptional control of the lac 
operon promoter. This plasmid, when present in E. coli, will direct the 
synthesis of methioninie-rennin. 
9. A Method of Obtaining Expression of Pre-Prorennin, Prorennin, and Rennin 
in Saccharomyces cerevisiae 
These three species, pre-prorennin, methionine-prorennin and 
methionine-valine-rennin may be expressed in S. cerevisiae using the 
promoter and other transcriptional and translational control regions from 
the S. cerevisiae uracil 3 gene. The yeast uracil 3 gene was placed on a 
plasmid (a shuttle vector which can be selected for and maintained in 
yeast or E. coli) in a form such that a truncated version of the 
.beta.-galactosidase or lac Z gene (missing 22 bp from its 5' end) is 
fused to the 3' end of a fragment of the ura 3 gene (missing about 900 bp 
from its 3' end). This is the Class III deletion #35 reported by M. Ross, 
M. J. Casadaban, and D. B. Botstein in Proc. Nat. Acad. Sci. USA 78 
2460-2464 (1981). On this plasmid, expression of the .beta.-galactosidase 
activity in yeast is under control of the uracil 3 gene control regions. 
We will use this deletion #35 to obtain expression of pre-prorennin, 
methionine-prorennin, and methionine-valine-rennin in S. cerevisiae as 
follows. 
First, a more complete deletion of the uracil 3 coding sequence will be 
obtained by cutting open DNA from deletion #35 with restriction 
endonuclease BamHI which cuts at the ura3-lacZ junction. This DNA will be 
resected with the nuclease Bal31 such that an average of 200 bp are 
removed. Next, BamHI synthetic oligonucleotide linkers (CRI) will be 
ligated onto the ends and the DNA will be ligated together so that a 
population of plasmids exists with BamHI sites at varying distances from 
the uracil control region. Gel electrophoresis of restriction-cut purified 
plasmid DNA will reveal a plasmid pCGS210 which contains very little ura3 
coding sequences. Sequencing of the BamHI site by the method of Maxam and 
Gilbert will confirm this. DNA from such a suitable Bal-resected cloned 
plasmid will be purified. This DNA will carry E. coli (ampicillin 
resistance) and yeast (Leu2 prototrophy) selectable markers, E. coli and 
yeast origins of replication, all of these being from plasmid pRB45 (M. 
Rose, M. J. Casadaban, D. Botstein, see above), and the ura3 control 
region with less than 50 nucleotides of ura3 coding material. In 
particular, one of these plasmids which we call pCGS210 will carry only 
10-20 nucleotides of ura3 coding material as determined by DNA sequencing 
by the method of Maxam and Gilbert. 
DNA coding for the 5' end of the pre-prorennin, prorennin and rennin will 
be obtained as follows. DNA coding for the pre-prorennin gene carrying the 
ATG translation initiatin codon and less than 20 nucleotides to the 5' 
side of the ATG will be obtained from the Bal31-resected rennin DNA-f1 
phage bank described in Section 8 above by screening those phage using 
restriction enzymes coupled with gel electrophoresis and sequencing by the 
Sanger method (F. Sanger et al J. Mol. Biol. 143 161-178 [1981]). DNA 
coding for prorennin with an ATG translation initiation codon will be 
obtained from plasmid pCGE181 described in Section 7 above. DNA coding for 
rennin with the ATG codon for translation initiation plus a GTC valine 
codon (phage 293-207-101) will be obtained from the rennin DNA-f1 phage 
blank as described in Section 8a above, or DNA coding for 
methionine-rennin will be obtained from plasmid pCGE304 described in 
Section 8b above. 
In order to obtain expressin in yeast of each of these pieces of DNA, the 
following experiments may be performed. The DNA coding for pre-prorennin, 
met-prorennin, met-val-rennin or met-rennin will be cut out of the 
appropriate phage or plasmid described above. The piece will be further 
cut with SmaI, and the desired fragment coding for a form of rennin will 
be purified by gel electrophoresis. Similarly, a BamHI (blunted) to SalI 
piece of DNA coding for the 'ZYA segment from E. coli (the gene for 
.beta.-galactosidase missing 22 bp from its 5' end plus the genes for 
lactose permease and lactose transacetylase) will be isolated from pRB45 
(M. Rose, M. J. Casadaban, and D. Botstein [19819 see above). Next, the 
plasmid pCGS210 described above will be cut at the unique BamHI site and 
resected for short distances with Bal31 nuclease (e.g. using the 
conditions of L. Guarente, G. Lauer, M. Ptashne [1980] see above) to yield 
a piece which has lost enough DNA toremove all the remaining ura3 coding 
sequences but not the control sequences. This DNA will also be cut with 
SalI, and the largest fragment will be gel purified. A trimolecular 
ligation reaction will be carried out using this vector fragment plus the 
BamHI (blunt) to SalI piece from pRB45 plus either the pre-prorennin 
met-prorennin or met-val-rennin DNA which was cut with SmaI and gel 
purified. A portion of this ligation reaction will be used to transform E. 
coli strain CGE4, and red colonies on MacConkey lactose plus amplicillin 
plates will be picked. Isolation and restriction enzyme analysis of the 
plasmid DNA will confirm the structure of the desired plasmids. 
Transformation into yeast strain CGY 80 (see below), selecting for leucine 
prototrophy and screening for blue color on minimal plus uracil (excess, 
for limiting amounts) plus leucine plus X-gal 
(5-Bromo-4-Chloro-3-indolyl-62-D-galactoside, Bachem, Calif.) medium will 
indicate that the plasmid directs translation of the appropriate 
rennin-.beta.-galactosidase fusion protein under uracil control. Finally, 
the .beta.-galactosidase coding portion of the desired plasmid will be 
removed by cutting the plasmid with BglII and SalI, the gel purifying the 
largest fragment. This piece will be ligated to the BglII to Hind III 
(which has been converted to a SalI site with synthetic oligonucleotide 
linkers CRI) fragment from phage 293-118/37 to regenerate the complete 
pre-prorennin, prorennin or rennin gene. These plasmids will direct 
translation of pre-prorennin, met-prorennin, met-val-rennin or met-rennin 
in the yeast S. cerevisiae. 
10. Expression of a Prorennin Fusion Protein in Yeast 
Due to the ready availability of a plasmid pRB71 (M. Rose and D. Botstein, 
submitted for publication) which resembles the deletion #35 of ura3 
described above (and in M. Rose, M. J. Casadaban and D. Botstein, 1981, 
Proc. Nat. Acad. Sci. USA 78 2460-2464) except only 11 nucleotides of ura3 
coding material remain before the BamHI site and the lactose operon ZYA 
genetic material, we have constructed a plasmid pCGS28 which carries the 
gene for prorennin fused to the 11 nucleotides of ura3 coding material 
such that a fusion protein will be synthesized in S. cerevisiae. This 
fusion protein consists of the authentic prorennin molecule, except the 
first four amino acids of prorennin have been replaced by 
methionine-serine-lysine-alanine. Activation to produce rennin results in 
the loss of the first 42 amino acids of prorennin so these initial four 
amino acids should have no effect on the final rennin product. The details 
of this plasmid construction are described below. 
In order to obtain efficient expression of prorennin in yeast, the ura3 
gene promoter region was used. This sequence of DNA has been cloned and is 
available on a plasmid (M. Rose, M. J. Casadaban and D. Botstein, 1981, 
Proc. Nat. Acad. Sci. USA 78, 2460-2464). The plasmid pRB72, obtained from 
M. Rose, bears the ura3 promoter region plus eleven nucleotides of the 
uracil 3 gene fused to a fragment of the lacZ gene missing the first 22 
nucleotides. The junction between the two incomplete genes is a BamHI 
restriction endonuclease site. This plasmid also contains the EcoRI A 
fragment from the 2.mu. plasmid of yeast, the leu2 gene from yeast, and 
the origin of replication plus the ampicillin resistance gene from pBR322, 
as described by M. Rose et al (see above). Thus, the plasmid can be grown 
and its presence can be selected for in either E. coli or S. cerevisiae. 
In order to obtain expression of a prorennin fusion protein (i.e., fused to 
the first 11 nucleotides of the ura3 gene and controlled by the ura3 
promoter) in yeast, two basic plasmid constructions were generated. The 
first is a ura3-prorennin-lacz fusion which when placed in yeast yields an 
active .beta.-galactosidase fusion protein, indicating that the ura3 
promoter is directing transcription of the desired fused genes. The second 
replaces the lacZ portion with the remainder of prorennin and results in 
yeast cells which produce a prorennin molecule bearing four amino acids 
specified by the ura3 gene. 
In the first construction, the 5' portion of the prorennin gene was 
inserted into the BamHI site of pRB71 such that ura3, prorennin, and lacZ 
are all in the same translational reading frame. This was accomplished as 
follows. Double-stranded recombinant f1 phage 293-118/37 DNA (12 .mu.g) 
bearing the entire prorennin gene was cut with 7 units of SmaI restriction 
endonuclease (N. E. Biolabs) for 2 hours at 37.degree. C. in a 50 .mu.l 
reaction containing 20 mM KCl, 6 mM Tris.HCl pH 8, 6 mM MgCl.sub.2 and 6 
mM 2-mercaptoethanol. The DNA was phenol extracted, ether extracted, and 
ethanol precipitated. Next, 250 pmoles of BamHI synthetic oligonucleotide 
linker (CRI, CCGGATCCGG), which had been phosphorylated at the 5' end 
using T.sub.4 polynucleotide kinase as described in Section 7 above, were 
ligated at 14.degree. C. overnight to the BamHI-cut phage DNA in a 40 
.mu.l reaction containing Buffer D and 900 units T.sub.4 DNA ligase (N.E. 
Biolabs). Following ligation, the reaction was diluted with five volumes 
of buffer containing 180 mM NaCl, 7 mM MgCl.sub.2 and 5 mM Tris.HCl pH 8, 
heated at 65.degree. C. for 5 minutes, chilled on ice and subjected to 
digestion for 3 hours with 15 units of BamHI endonuclease added ech hour. 
After phenol extraction, ether extraction, and ethanol precipitation, the 
DNA was redissolved in water and subjected to electrophoresis in a 2% 
agarose gel. DNA was eluted from the band corresponding to the 
approximately 440 bp BamHI-SmaI(BamHI-linkered) fragment by macerating the 
frozen gel piece and collecting the residual liquid (freeze-thaw method). 
The DNA fragment was ethanol precipitated and redissolved in 6 .mu.l 
water. About 5 .mu.g of plasmid pRB71 DNA was digested with 20 units BamHI 
endonuclease (N.E. Biolabs) for 2 hours at 37.degree. C. in a 50 .mu.l 
reaction containing Buffer X plus 6 mM 2-mercapto ethanol. After phenol 
extraction, ether extraction, and ethanol precipitation, the DNA was 
redissolved in 20 .mu.l water and treated with 0.1 unit calf intestinal 
alkaline phosphatase (Boehringer/Mannheim) for 30 minutes at 37.degree. C. 
in a 50 .mu.l reaction containing Buffer C. The phenol extracted, ethanol 
precipitated DNA was redissolved in 6 .mu.l water and added to a ligation 
reaction containing 6 .mu.l of the BamHI-SmaI (BamHI-linkered) fragment, 
and the ligation was carried out with 600 units of T.sub.4 DNA ligase 
(N.E. Biolabs) at 14.degree. C. overnight in 20 .mu.l of Buffer L. Cells 
of E. coli strain CGE6 were transformed with the ligated DNA and 
ampicillin resistant transformants were obtained as described above. About 
200 transformants were tested by the colony hybridization method of M. 
Grunstein and D. S. Hogness (1975, Proc. Nat. Acad. Sci. USA, 72, 
3961-3965) using as probe .alpha.-.sup.32 P-labelled nick-translated 
recombinant phage 293-207 DNA. Almost 20% of the transformants contained 
rennin sequences by this criteria. Plasmid DNA was prepared from ten of 
the transformants, and the orientation of the insert was determined from 
the pattern of fragments produced by digestion with PstI endonuclease. One 
of the plasmids, pCGS16, which contained the prorennin fragment in the 
proper orientation was used to transform S. cerevisiae strain CGY80 (MaTa, 
leu2-3, leu2-112, his3, trp1-289, ura3-52) according to the protocol of A. 
Hinnen, J. B. Hicks and G. Fink (1978, Proc. Nat. Acad. Sci. USA 75, 
1929-1933). Yeast transformants which were capable of growth without added 
leucine due to the presence of leu2 gene on the plasmid, were streaked 
onto minimal medium plates containing the chromogenic substrate X-gal 
(exactly as described by M. Rose et al see above) and supplemented with 
uracil, tryptophan and histidine. All of the transformants examined 
produced blue colonies on the X-gal minimal medium indicating that 
.beta.-galactosidase is produced. This means that the ura3-prorennin-lacZ 
fusion protein is produced and that the translational reading frame for 
each of the three protein fragments is the same. 
This result suggested that a similar plasmid should direct the expression 
of a ura3-prorennin fusion protein if the .beta.-galactosidase sequences 
are replaced with the remainder of the prorennin gene. Accordingly, 8 
.mu.g of plasmid pCGS16 were digested with 5 units of BglII restriction 
endonuclease (N.E. Biolabs) for one hour at 37.degree. C. in 80 .mu.l of 
Buffer P. Next, 10 .mu.l 1M NaCl and 6 .mu.l water was added to the 
reaction and the DNA was further digested with 16 units SalI endonuclease 
for one hour at 37.degree. C. After phenol extraction, ether extraction, 
and ethanol precipitation, the DNA was treated with 0.06 units calf 
intestinal alkaline phosphatase (Boehringer/Mannheim) for 15 minutes at 
37.degree. C. in 50 .mu.l containing Buffer C. The reaction was terminated 
by phenol extraction of the DNA and ethanol precipitation. 
Meanwhile, about 15 .mu.g of recombinant f1 phage 293-118/37 
double-stranded DNA was cut with 12 units of Hind III (N.E. Biolabs) for 2 
hours at 37.degree. C. in 100 .mu.l of Buffer H. After phenol and ether 
extraction and ethanol precipitation, the DNA (6 .mu.g) was rendered 
blunt-ended by treatment with 10 units E. coli DNA polymerase 
(Boehringer/Mannheim) for 10 minutes at 10.degree. C. in 40 .mu.l Buffer 
D. Next, 250 pmoles of SalI synthetic oligonucleotide linker (CRI, 
GGTCGACC) which had been phosphorylated using T.sub.4 polynucleotide 
kinase as described above was added along with sufficient buffer 
components to keep the concentration of all components constant. The 
linkers were ligated onto the DNA by incubation with 900 units of T.sub.4 
DNA Ligase (N.E. Biolabs) at 14.degree. C. overnight. Next, five volumes 
of buffer consisting of 10 mM Tris.HCl pH 8, 10 mM MgCl.sub.2 and 180 mM 
NaCl was added, the solution heated at 65.degree. C. for 5 minutes, 
chilled on ice and then incubated for 5 hours at 37.degree. C. with an 
addition of 20 units of SalI restriction endonuclease (N.E. Biolabs) each 
hour. After the DNA was phenol extracted, ether extracted, and ethanol 
precipitated, it was redissolved in 20 .mu.l water and digested with 5 
units BglII (N.E. Biolabs) for one hour 37.degree. C. in a 30 .mu.l volume 
containing Buffer P. Then the reaction was terminated with 1/10 volume of 
200 mM EDTA and applied to a 2% agarose gel. The band corresponding to the 
approximately 1000 bp BglII-Hind III (SalI-linkered) fragment was excised 
and the DNA was recovered by the freeze-thaw method described above. 
The ethanol precipitated pCGS16 DNA which had been cut with BglII and SalI 
endonuclease was redissolved in 13 .mu.l water along with the gel purified 
293-118/37 BglII-Hind III (SalI-linkered) DNA fragment and the two pieces 
were ligated together in a 20 .mu.l reaction containing Buffer L and 600 
units T.sub.4 DNA ligase (N.E. Biolabs). Cells of strain CGE6 were treated 
with CaCl.sub.2 and transformed with the ligated DNA as described above. 
Plasmid DNA was purified from five different ampicillin-resistant 
transformants and subjected to digestion with BamHI or PstI plus SalI. The 
positions of the bands in a 2% agarose gel indicated that the entire 
prorennin sequence is present in plasmid pCGS28. 
Accordingly, the yeast strain CGY80 was transformed with the plasmid DNA by 
the method of A. Hinnen, J. B. Hicks, and G. Fink (1978, see above) and 
leucine prototrophs were selected. One such transformant, CGY116, was 
grown to exponential phase in minimal medium containing the appropriate 
amino acid supplements, labelled with 100 .mu.Ci .sup.35 S-L-methionine 
for one-half generation at 30.degree. and lysed by vortexing with glass 
beads (250-300 .mu.m) for 3 minutes. The extract was clarified by 
centrifugation and immunoprecipitated with rennin antiserum. The 
immunoprecipitate was dissolved in SDS sample buffer and subjected to 
electrophoresis in a 10% polyacrylamide gel containing 0.1% SDS according 
to the method of U. K. Laemmli and M. Favre (see above). Autoradiography 
revealed that strain CGY116 carrying the plasmid pCGS28 directs the 
synthesis of a protein which reacts with rennin antiserum and is the size 
expected for prorennin. Furthermore, excess unlabelled rennin present 
during the immunoprecipitation eliminates the radioactive band otherwise 
present in the prorennin position. Therefore, S. cerevisiae strain CGY116 
produces calf prorennin fused to four amino acids from the yeast ura3 
gene in place of the first four amino acids of prorennin. Activation of 
the ura3-prorennin fusion protein by standard methods described by B. 
Foltman (Methods in Enzymology 19 421-436, 1970) should yield active 
rennin identical to authentic calf rennin A since the "pro" zymogen 
peptide (including the four "foreign" amino acids in this case) will be 
cleaved off during activation. Strain CGY 116 bearing plasmid pCGS28 is on 
deposit with the American Type Culture Collection (ATCC) and its Accession 
number is 20623. 
Strain CGY116 is S. cerevisiae strain CGY80 (MAT a, leu 2-3, leu2-112, 
ura3-52, his 3.gradient., trp 1-289, carrying the plasmid pCGS 28, which 
is defined as follows (and see FIG. 6 below). The plasmid contains most of 
plasmid pBR322 (J. G. Sutcliffe [1979] Cold Spring Harbor Symposium 43, 
77-90), the Eco RI, A fragment of the yeast 2.mu. plasmid (J. L. Hartley 
and J. E. Donelson [1980] Nature 286, 860-865), the Sal I-Xho I fragment 
of yeast chromosomal DNA carrying the LEU 2 gene (A. Hinnen, J. Hicks and 
G. R. Fink [1978] Proc. Nat. Acad. Sci. USA, 75 1929-1933), a fragment 
from the yeast chromosomal DNA consisting of a portion of the ura3 gene 
(from BamHI to a site 11 nucleotides 3' to the initiation of translation 
as in pRB71 plus the prorennin A gene from the BamHI site at nucleotide 
#267 to the end of the gene. 
With reference again to Table 1, the nucleotide and amino acid sequence of 
preprorennin A as shown, illustrates the nucleotide sequences for several 
of the materials of this invention. These materials can be cut from the 
nucleotide sequence shown by conventional procedures. Similarly the 
pre-prorennin A form can be changed to the prorennin B form by 
substituting a glycine residue at position number 290 in place of the 
aspartate residue at this position. Useful products obtained fom the 
pre-prorennin A derived by the process of this invention as shown in Table 
1 include the following nucleotide sequences forming part of the 
recombinant DNA material: 
1. A gene coding for a polypeptide displaying rennin activity having a 
nucleotide sequence as shown from numbers 379 to 1350 in Table 1 and 
repeated below. 
3 
nucleotide sequence as shown from numbers 379 to 1350 in Table 1 and 
repeated below. 
GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GAT AGT CAG TAC TTT 
GGG AAG ATC GLY GLU VAL ALA SER VAL PRO LEU THR ASN TYR LEU ASP SER GLN 
TYR PHE GLY LYS ILE TAC CTC GGG ACC CCG CCC CAG GAG TTC ACC GTG CTG TTT 
GAC ACT GGC TCC TCT GAC TTC TYR LEU GLY THR PRO PRO GLN GLU PHE THR VAL 
LEU PHE ASP THR GLY SER SER ASP PHE TGG GTA CCC TCT ATC TAC TGC AAG AGC 
AAT GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG TRP VAL PRO SER ILE TYR CYS 
LYS SER ASN ALA CYS LYS ASN HIS GLN ARG PHE ASP PRO AGA AAG TCG TCC ACC 
TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC CAC TAC GGG ACA GGC ARG LYS SER 
SER THR PHE GLN ASN LEU GLY LYS PRO LEU SER ILE HIS TYR GLY THR GLY AGC 
ATG CAG GGC ATC CTG GGC TAT GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC 
CAG SER MET GLN GLY ILE LEU GLY TYR ASP THR VAL THR VAL SER ASN ILE VAL 
ASP ILE GLN CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GTC TTC ACC 
TAT GCC GAA TTC GAC GLN THR VAL GLY LEU SER THR GLN GLU PRO GLY ASP VAL 
PHE THR TYR ALA GLU PHE ASP GGG ATC CTG GGG ATG GCC TAC CCC TCG CTC GCC 
TCA GAG TAC TCG ATA CCC GTG TTT GAC GLY ILE LEU GLY MET ALA TYR PRO SER 
LEU ALA SER GLU TYR SER ILE PRO VAL PHE ASP AAC ATG ATG AAC AGG CAC CTG 
GTG GCC CAA GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT ASN MET MET ASN ARG 
HIS LEU VAL ALA GLN ASP LEU PHE SER VAL TYR MET ASP ARG ASN GGC CAG GAG 
AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC TAC ACA GGG TCC CTG GLY 
GLN GLU SER MET LEU THR LEU GLY ALA ILE ASP PRO SER TYR TYR THR GLY SER 
LEU CAC TGG GTG CCC GTG ACA GTG CAG CAG TAC TGG CAG TTC ACT GTG GAC AGT 
GTC ACC ATC HIS TRP VAL PRO VAL THR VAL GLN GLN TYR TRP GLN PHE THR VAL 
ASP SER VAL THR ILE AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GCC 
ATC CTG GAC ACG GGC ACC TCC SER GLY VAL VAL VAL ALA CYS GLU GLY GLY CYS 
GLN ALA ILE LEU ASP THR GLY THR SER AAG CTG GTC GGG CCC AGC AGC GAC ATC 
CTC AAC ATC CAG CAG GCC ATT GGA GCC ACA CAG LYS LEU VAL GLY PRO SER SER 
ASP ILE LEU ASN ILE GLN GLN ALA ILE GLY ALA THR GLN AAC CAG TAC GAT GAG 
TTT GAC ATC GAC TGC GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC ASN GLN TYR 
ASP GLU PHE ASP ILE ASP CYS ASP ASN LEU SER TYR MET PRO THR VAL VAL TTT 
GAG ATC AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT ACC AGC CAG GAC 
CAG PHE GLU ILE ASN GLY LYS MET TYR PRO LEU THR PRO SER ALA TYR THR SER 
GLN ASP GLN GGC TTC TGT ACC AGT GGC TTC CAG AGT GAA AAT CAT TCC CAG AAA 
TGG ATC CTG GGG GAT GLY PHE CYS THR SER GLY PHE GLN SER GLU ASN HIS SER 
GLN LYS TRP ILE LEU GLY ASP GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC 
AGG GCC AAC AAC CTC GTG GGG CTG GCC VAL PHE ILE ARG GLU TYR TYR SER VAL 
PHE ASP ARG ALA ASN ASN LEU VAL GLY LEU ALA AAA GCC ATC TGA LYS ALA 
ILE 
2. A gene coding for a polypeptide displaying pre-prorennin activity having 
a nucleotide sequence as shown from numbers 205-1350 in Table 1 and 
repeated below. 
3 
2. A gene coding for a polypeptide displaying pre-prorennin activity 
having a nucleotide sequence as shown from numbers 205-1350 in Table 1 
and repeated below. 
ATG AGG TGT CTC GTG GTG CTA CTT GCT GTC TTC GCT CTC TCC CAG GGC 
MET ARG CYS LEU VAL VAL LEU LEU ALA VAL PHE ALA LEU SER GLN GLY GCT GAG 
ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TCT CTG AGG AAG GCG CTG AAG GAG 
CAT ALA GLU ILE THR ARG ILE PRO LEU TYR LYS GLY LYS SER LEU ARG LYS ALA 
LEU LYS GLU HIS GGG CTT CTG GAG GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC 
AGC AGC AAG TAC TCC GGC TTC GLY LEU LEU GLU ASP PHE LEU GLN LYS GLN GLN 
TYR GLY ILE SER SER LYS TYR SER GLY PHE GGG GAG GTG GCC AGC GTG CCC CTG 
ACC AAC TAC CTG GAT AGT CAG TAC TTT GGG AAG ATC GLY GLU VAL ALA SER VAL 
PRO LEU THR ASN TYR LEU ASP SER GLN TYR PHE GLY LYS ILE TAC CTC GGG ACC 
CCG CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TCT GAC TTC TYR LEU 
GLY THR PRO PRO GLN GLU PHE THR VAL LEU PHE ASP THR GLY SER SER ASP PHE 
TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT GCC TGC AAA AAC CAC CAG CGC TTC 
GAC CCG TRP VAL PRO SER ILE TYR CYS LYS SER ASN ALA CYS LYS ASN HIS GLN 
ARG PHE ASP PRO AGA AAG TCG TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT 
ATC CAC TAC GGG ACA GGC ARG LYS SER SER THR PHE GLN ASN LEU GLY LYS PRO 
LEU SER ILE HIS TYR GLY THR GLY AGC ATG CAG GGC ATC CTG GGC TAT GAC ACC 
GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG SER MET GLN GLY ILE LEU GLY TYR 
ASP THR VAL THR VAL SER ASN ILE VAL ASP ILE GLN CAG ACA GTA GGC CTG AGC 
ACC CAG GAG CCC GGG GAC GTC TTC ACC TAT GCC GAA TTC GAC GLN THR VAL GLY 
LEU SER THR GLN GLU PRO GLY ASP VAL PHE THR TYR ALA GLU PHE ASP GGG ATC 
CTG GGG ATG GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GTG TTT GAC 
GLY ILE LEU GLY MET ALA TYR PRO SER LEU ALA SER GLU TYR SER ILE PRO VAL 
PHE ASP AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA GAC CTG TTC TCG GTT TAC 
ATG GAC AGG AAT ASN MET MET ASN ARG HIS LEU VAL ALA GLN ASP LEU PHE SER 
VAL TYR MET ASP ARG ASN GGC CAG GAG AGC ATG CTC ACG CTG GGG GCC ATC GAC 
CCG TCC TAC TAC ACA GGG TCC CTG GLY GLN GLU SER MET LEU THR LEU GLY ALA 
ILE ASP PRO SER TYR TYR THR GLY SER LEU CAC TGG GTG CCC GTG ACA GTG CAG 
CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC HIS TRP VAL PRO VAL THR 
VAL GLN GLN TYR TRP GLN PHE THR VAL ASP SER VAL THR ILE AGC GGT GTG GTT 
GTG GCC TGT GAG GGT GGC TGT CAG GCC ATC CTG GAC ACG GGC ACC TCC SER GLY 
VAL VAL VAL ALA CYS GLU GLY GLY CYS GLN ALA ILE LEU ASP THR GLY THR SER 
AAG CTG GTC GGG CCC AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GCC 
ACA CAG LYS LEU VAL GLY PRO SER SER ASP ILE LEU ASN ILE GLN GLN ALA ILE 
GLY ALA THR GLN AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC GAC AAC CTG AGC 
TAC ATG CCC ACT GTG GTC ASN GLN TYR ASP GLU PHE ASP ILE ASP CYS ASP ASN 
LEU SER TYR MET PRO THR VAL VAL TTT GAG ATC AAT GGC AAA ATG TAC CCA CTG 
ACC CCC TCC GCC TAT ACC AGC CAG GAC CAG PHE GLU ILE ASN GLY LYS MET TYR 
PRO LEU THR PRO SER ALA TYR THR SER GLN ASP GLN GGC TTC TGT ACC AGT GGC 
TTC CAG AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT GLY PHE CYS THR 
SER GLY PHE GLN SER GLU ASN HIS SER GLN LYS TRP ILE LEU GLY ASP GTT TTC 
ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AAC AAC CTC GTG GGG CTG GCC 
VAL PHE ILE ARG GLU TYR TYR SER VAL PHE ASP ARG ALA ASN ASN LEU VAL GLY 
LEU ALA AAA GCC ATC TGA LYS ALA ILE 
3. A gene coding for a polypeptide displaying prorennin activity having a 
nucleotide sequence as shown from numbers 253-1350 in Table 1 and repeated 
below. 
3 
3. A gene coding for a polypeptide displaying prorennin activity having 
a nucleotide sequence as shown from numbers 253-1350 in Table 1 and 
repeated below. 
GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TCT CTG AGG AAG GCG 
CTG AAG GAG CAT ALA GLU ILE THR ARG ILE PRO LEU TYR LYS GLY LYS SER LEU 
ARG LYS ALA LEU LYS GLU HIS GGG CTT CTG GAG GAC TTC CTG CAG AAA CAG CAG 
TAT GGC ATC AGC AGC AAG TAC TCC GGC TTC GLY LEU LEU GLU ASP PHE LEU GLN 
LYS GLN GLN TYR GLY ILE SER SER LYS TYR SER GLY PHE GGG GAG GTG GCC AGC 
GTG CCC CTG ACC AAC TAC CTG GAT AGT CAG TAC TTT GGG AAG ATC GLY GLU VAL 
ALA SER VAL PRO LEU THR ASN TYR LEU ASP SER GLN TYR PHE GLY LYS ILE TAC 
CTC GGG ACC CCG CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TCT GAC 
TTC TYR LEU GLY THR PRO PRO GLN GLU PHE THR VAL LEU PHE ASP THR GLY SER 
SER ASP PHE TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT GCC TGC AAA AAC CAC 
CAG CGC TTC GAC CCG TRP VAL PRO SER ILE TYR CYS LYS SER ASN ALA CYS LYS 
ASN HIS GLN ARG PHE ASP PRO AGA AAG TCG TCC ACC TTC CAG AAC CTG GGC AAG 
CCC CTG TCT ATC CAC TAC GGG ACA GGC ARG LYS SER SER THR PHE GLN ASN LEU 
GLY LYS PRO LEU SER ILE HIS TYR GLY THR GLY AGC ATG CAG GGC ATC CTG GGC 
TAT GAC ACC GTC ACT GTC TCC ACC ATT GTG GAC ATC CAG SER MET GLN GLY ILE 
LEU GLY TYR ASP THR VAL THR VAL SER ASN ILE VAL ASP ILE GLN CAG ACA GTA 
GGC CTG AGC ACC CAG GAG CCC GGG GAC GTC TTC ACC TAT GCC GAA TTC GAC GLN 
THR VAL GLY LEU SER THR GLN GLU PRO GLY ASP VAL PHE THR TYR ALA GLU PHE 
ASP GGG ATC CTG GGG ATG GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC 
GTG TTT GAC GLY ILE LEU GLY MET ALA TYR PRO SER LEU ALA SER GLU TYR SER 
ILE PRO VAL PHE ASP AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA GAC CTG TTC 
TCG GTT TAC ATG GAC AGG AAT ASN MET MET ASN ARG HIS LEU VAL ALA GLN ASP 
LEU PHE SER VAL TYR MET ASP ARG ASN GGC CAG GAG AGC ATG CTC ACG CTG GGG 
GCC ATC GAC CCG TCC TAC TAC ACA GGG TCC CTG GLY GLN GLU SER MET LEU THR 
LEU GLY ALA ILE ASP PRO SER TYR TYR THR GLY SER LEU CAC TGG GTG CCC GTG 
ACA GTG CAG CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC HIS TRP VAL 
PRO VAL THR VAL GLN GLN TYR TRP GLN PHE THR VAL ASP SER VAL THR ILE AGC 
GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GCC ATC CTG GAC ACG GGC ACC 
TCC SER GLY VAL VAL VAL ALA CYS GLU GLY GLY CYS GLN ALA ILE LEU ASP THR 
GLY THR SER AAG CTG GTC GGG CCC AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC 
ATT GGA GCC ACA CAG LYS LEU VAL GLY PRO SER SER ASP ILE LEU ASN ILE GLN 
GLN ALA ILE GLY ALA THR GLN AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC GAC 
AAC CTG AGC TAC ATG CCC ACT GTG GTC ASN GLN TYR ASP GLU PHE ASP ILE ASP 
CYS ASP ASN LEU SER TYR MET PRO THR VAL VAL TTT GAG ATC AAT GGC AAA ATG 
TAC CCA CTG ACC CCC TCC GCC TAT ACC AGC CAG GAC CAG PHE GLU ILE ASN GLY 
LYS MET TYR PRO LEU THR PRO SER ALA TYR THR SER GLN ASP GLN GGC TTC TGT 
ACC AGT GGC TTC CAG AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT GLY 
PHE CYS THR SER GLY PHE GLN SER GLU ASN HIS SER GLN LYS TRP ILE LEU GLY 
ASP GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AAC AAC CTC GTG 
GGG CTG GCC VAL PHE ILE ARG GLU TYR TYR SER VAL PHE ASP ARG ALA ASN ASN 
LEU VAL GLY LEU ALA AAA GCC ATC TGA LYS ALA ILE 
4. A pre-prorennin signal sequence coding for sixteen amino acids including 
an initiator ATG codon comprising nucleotides 205-252 of Table 1. This 
sequence directs the secretion of pre-prorennin from stomach cells which 
synthesize it, and hence it is believed useful when attached to other 
cloned genes for directing secretion of their protein products out of host 
cells into a periplasmic space or into culture media. In addition, these 
extra nucleotides, not required for rennin activity, when translated into 
amino acids may play a role in stabilizing the enzyme against proteolytic 
degradation while it is inside the cell. This could be of general 
usefulness for stabilizing the cloned gene products in various host cells 
and in shelf items. 
5. A "pro" or zymogen sequence at nucleotide Nos. 253-378 in Table 1 which 
is a sequence of 126 nucleotides coding for 42 amino acids which form the 
zymogen portion of the prorennin molecule. This sequence forms the 
inactive zymogen of rennin and is removed to generate active rennin. The 
inactive zymogen can have long shelf life. It may also stabilize the 
rennin molecule and thus may be of general usefulness for stabilizing gene 
products of other cloned genes. 
As used herein, the term "genetic material derived from recombinant DNA 
material" indicates the genetic material of the host cells which have been 
transformed with recombinant DNA and cloned to obtain cells which carry 
the genetic information for the desired product. Recombinant DNA material 
is used in its normal sense to denote DNA derived from two or more 
different sources joined or spliced together to form a single molecule but 
also includes synthesized DNA obtained for example by chemical synthesis. 
Obviously the recombinant methods used to isolate and obtain the original 
recombinant DNA material may produce host cells which are then cloned and 
grown without the need for reuse of genetic recombinant methods. In such 
case, the cloned cells are considered to be derived from the cells which 
were originally treated by recombinant DNA methods and are considered to 
contain genetic material derived from recombinant DNA material. 
As described above, recombinant DNA molecules are formed comprising genes 
coding for at least one polypeptide displaying milk clotting activity or 
useful in producing such polypeptides. 
Although specific prorennin, pre-prorennin and rennin genes are 
specifically set forth in Table 1, it should be understood that these 
terms as used herein include functional equivalents thereof having any 
changes in nucleotide or amino acid sequences or alterations which do not 
significantly affect milk clotting or catalytic activity of the final 
rennin product. Thus the broad term "rennin" as used in rennin, 
pre-prorennin and prorennin is meant to include any sequence of amino 
acids that clots mammalian milk such as bovine or goat's milk, and thus 
may include selected fragments of rennin as previously sequenced in the 
prior art. The rennin, pre-prorennin and prorennin can have non-functional 
amino acid sequences attached thereto which can be removed by conventional 
methods to enhance the desired activity of the polypeptide. 
As mentioned above, calf rennin exists in two allelic forms A and B which 
differ at the 290 amino acid sequence position and possibly at the 204 
position. Although the cloning and expression of rennin A is described 
here, a gene for rennin B may be readily generated from the A form gene by 
simple techniques outlined below. Expression of rennin B may be obtained 
in a manner identical to that described here for the A form. In order to 
generate a gene for rennin B, oligonucleotides spanning the regions which 
are to be changed and including the desired changes could be chemically 
synthesized. For example, two 20-mer oligonucleotides, one of sequence 
identical to nucleotides 847-866 (Table 1) except nucleotide 856 is 
changed from an A to a G, and one of sequence identical to nucleotides 
1099-1118 (Table 1) except nucleotide 1109 is changed from A to G, would 
be synthesized and used to prime second strand DNA synthesis off of fl 
phage 293-118/37 double-stranded DNA which had been randomly nicked and 
converted to single strands with endonuclease III by the method of R. B. 
Wallace et al (Science [1980] 209 1396-1400). The resulting 
double-stranded circular DNA would be ligated with T.sub.4 DNA polymerase 
and used to transform an appropriate E. coli strain. A mixture of phages 
will result, some carrying the gene for renning A and some carrying the 
modified rennin A gene bearing one or the other of the two specified 
changes. DNA sequencing of the relevant restriction fragments will allow 
selection of phage carrying each desired changes and a complete rennin B 
gene may be generated by splicing the two together at an appropriate 
restriction site. If the rennin A to be converted to rennin B differs from 
rennin B only at the 290 position, then only the synthetic oligonucleotide 
spanning the region 1099 to 1118 need be used and the sequence for 847-866 
is not used. 
Although the methods of this invention describe starting with RNA material 
it is also possible to start by isolating the gene derived from the 
genomic DNA. In that case the intervening sequences would be first spliced 
out or a suitable host organism would be used which is capable of 
processing the RNA to remove intervening sequences. It is also possible to 
start by chemically synthesizing the appropriate RNA or DNA, or portions 
thereof, and then employing procedures described herein ultimately to 
obtain the expression of either rennin, pre-prorennin and/or prorennin. 
Furthermore, cloned genes for the milk-clotting proteins of other 
organisms, such as sheep, goat, pig or water buffalo, which may produce 
rennin-like enzymes can be generated using the procedures described here.