Expression systems for amidating enzyme

Alpha-amidating enzyme is produced by recombinant DNA techniques recoverable in high yields and at high purity. Both eukaryotic and prokaryotic expression vectors are provided having a transcriptional promoter followed downstream by a DNA sequence which encodes amidating enzyme. The vector selected is one capable of directing the expression of polypeptides in the host selected, and preferred hosts are transected with the described vectors.

BACKGROUND OF THE INVENTION 
This invention relates to the production of alpha-amidating enzymes through 
recombinant DNA techniques, and particularly to expression vectors and 
hosts capable of expressing alpha-amidating enzyme in high yields and at 
recoverable high purity. 
The intracellular processing (cleavage and/or functional group 
modification) of precursor forms of native proteins following their 
translation from nucleic acid coding sequences has been clearly 
documented. 
In general, mammalian cells and other eukaryotes can perform certain 
post-translational processing procedures, while prokaryotes cannot. 
Certain prokaryotes, such as E. coli, are widely employed as hosts for the 
production of mammalian proteins via recombinant DNA (rDNA) technology 
because they can be readily grown in batch fermentation procedures and 
because they are genetically well-characterized. However, many mammalian 
proteins require some type of post-translational processing, and if these 
proteins are produced by genetic engineering of E. coli, for example, the 
post-translational processing must often be accomplished by using complex, 
in vitro chemical procedures which are cost-prohibitive for large-scale 
production applications. 
One type of processing activity involves the specific amidation of the 
carboxyl-terminal amino acid of a peptide or protein. Many 
naturally-occurring hormones and peptides contain such a modification, 
which is often essential if the protein is to be biologically active. An 
example is calcitonin, where the substitution of a non-amidated proline 
residue for the a-rideted proline of the native form results in a 
3,000-fold reduction in biological activity. Other biological peptides 
requiring post-translational amidation for full activity include but are 
not limited to growth hormone releasing factor, other calcitonins. and 
calcitonin gene-related peptide. 
The specific amidation of the carboxyl-terminal amino acid of a protein is 
catalyzed by alpha-amidating enzymes. The polypeptide sequences for many 
important biological proteins which require amidaLion for maximal 
efficacy, may be manufactured, for example, by genetic engineering 
techniques. However, the important and sometimes essential carboxyl 
terminal amidation must often be performed in vitro. It is desirable to 
avoid costly and cumbersome chemical amidation techniques at this point, 
and is therefore desirable to utilize an amidating enzyme to perform the 
specific amidation. However, alpha-amidating enzyme is not easily obtained 
in nature. 
The presence of amidated peptides in a particular tissue is not necessarily 
synonymous with high levels of alpha-amidating enzyme. For example, rat 
anterior pituitary tissue contains high alpha-amidating activity but no 
known substrates Eipper et al, PNAS 80, 5144-5148 (1983)!. Rat posterior 
pituitary tissue contains amidated peptides (oxytocin and vasopressin) but 
has very little alpha-amidatirig activity Eipper et al., Endo 116, 
2497-2504 (1985)!. Therefore, until individual tissues are tested for 
alpha-amidating activity, the presence or potential levels of the enzyme 
cannot be anticipated. 
An even greater impediment to the availability of amidating enzyme obtained 
from natural sources is the usually low level of purity. Amidating enzymes 
obtainable from natural sources are contaminated with proteolytic enzymes 
and other impurities. Effective recovery of amidated product is greatly 
hindered when these impurity-laced enzymes are used to amidate a substrate 
comprised of L-amino acids. The presence of proteases, in particular, may 
break down the substrate and/or the amidated product and/or the amidating 
enzyme itself. Most biologically importaant polypeptides comprise L-amino 
acids, and are susceptable to this proteolytic breakdown and to other 
amidation-hindering Impediments caused by impurities in amidaring enzyme 
preparations. 
Because nature provides few sources, low abundance and insufficient purity 
of alpha-amidating enzyme, there is a need for efficient methods of mass 
producing alpha-amidating enzyme recoverable in high yield and at high 
purity. 
As used herein, the terms "amidating enzyme" and "alpha-amidating enzyme" 
refer to any agent capable of catalyzing the conversion of a peptidyl 
substrate to a corresponding peptidyl amide having an amino group in place 
of the C-terminal amino acid of said substrate. 
BRIEF DESCRIPTION OF THE INVENTION 
It is an object of the present invention to provide alpha-amidating enzyme 
recoverable in high yields and at high purity. 
It is another object of the invention to provide host organisms capable of 
expressing alpha-amidating enzymes recoverable in high yield and at high 
purity. 
It is another object of the invention to provide expression vectors 
containing DNA sequences coding for alpha-amidating enzyme. 
It is another object of the invention to provide expression vectors capable 
of expressing alpha-amidating enzyme in a manner wherein expressed enzyme 
may be easily recovered and purified to levels effective for amidation of 
peptidyl substrates comprising L-amino acids, for example, substrates 
purified from natural sources, synthesized chemically, or produced by 
recombinant DNA techniques. 
It is another object of the invention to provide expression vectors 
especially suited for directing the expression of alpha-amidating enzymes 
in a eukaryotic host. 
It is another object of the invention to provide expression vectors 
especially suited for directing the expression of aloha-amidating enzymes 
in a prokaryotic host. 
It is another object of the invention to provide a means for efficient 
cost-effective mass production of aipha-amidating enzyme. 
These and other objects are accomplished by providing a host capable of 
expressing a polypepotide sequence of an alpha amidating enzyme, said host 
comprising an expression vector which includes a transcriptional promoter 
followed downstream by a DNA sequence foreign to said host which encodes 
said amidating enzyme, said vector being capable of directing expression 
of polypepotides in said host. 
In certain embodiments, a host is provided which is capable of expressing 
the polypeptide sequence of an alpha amidating enzyme, said host 
comprising an expression vector containing a transcriptional promoter 
followed downstream by a DNA sequence foreign to said host which is 
capable of hybridizing under stringent conditions with a DNA sequence of 
FIGS. 5A-5F. 
In another embodiment, a host is provided which is capable of expressing 
the polypeptide sequence of an alpha amnidating enzyme, said host 
comprising an expression vector containing a transcriptional promoter 
followed downstream by a DNA sequence foreign to said host which is 
capable of hybridizing under stringent conditions with a DNA sequence of 
FIGS. 6A-6F. 
As used herein, the term "stringent conditions" means 2.times.SSC (0.3M 
sodium chloride and 0.03M sodium citrate) at 62.degree. C. 
The present invention also provides expression vectors for directing 
expression of alpha-amidating enzyme in both prokaryotic and eukaryotic 
systems. For example, an expression vector is provided which is capable of 
directing, in a prokaryotic host, the expression of a polypeptide sequence 
of an alpha amidating enzyme, said vector comprising a transcriptional 
promoter followed downstream by a first DNA sequence having an amidating 
enzyme-coding region, said first sequence being sufficiently homologous to 
a natural DNA sequence for expressing natural amidating enzyme to undergo 
hybridization with said natural sequence under stringent conditions, and 
said first sequence including an initiating methionine codon within about 
50 nucleotides of the start of said enzyme-coding region. 
Likewise, an expression vector is provided, in another embodiment of the 
invention, which is capable of directing the expression of a polypeptide 
sequence of an alpha-amidating enzyme in a eukaryotic host, said vector 
comprising a transcriptional promoter followed downstream by a first DNA 
sequence having an amidating enzyme-coding region, said first sequence 
being sufficiently homologous to a natural DNA sequence for expressing 
natural amidating enzyme to undergo hybridization with said natural 
sequence under stringent conditions, and said first sequence including a 
stop codon upstream from a sequence which would otherwise code for a 
membrane spanning domain. 
This first sequence should be followed by a sequence specifying the 
addition of poly A to the messenger RNA generated by transcription from 
said promoter. 
As used herein, the term "membrane spanning domain" is a DNA sequence 
which, as determined by the test of Kyte & Doolittle, J. Mol. Biol., Vol. 
157, pp. 105-132 (1982) (the entire disclosure of which is hereby 
incorporated by reference), codes for an amino acid sequence of sufficient 
hydrophobicity, length, structural character, and the like to become fixed 
in the membrane. For example, this may occur as a protein is synthesized 
on a membrane-bound ribosome or, alternatively, the amino acid sequence 
coded by the membrane spanning domain may become associated with other 
areas of the protein of which it is a part, such that the sequence becomes 
inserted into the hydrophobic environment of the membrane 
post-translationally. Membrane-spanning domains are discussed in more 
detail in Von Heine, Seauence Analysis in Molecular Biology: Treasure 
Trove or Trivial Pursuit, pp. 81-121 (Acad. Press 1987), the teachings of 
which are hereby incorporated by reference. 
The base numbers utilized herein are the numbers specifically stated for 
any DNA sequence expressly set forth together with base number references. 
For all sequences for which base numbers are not expressly assigned 
herein, the bases shall be consecutively numbered with base number 1 being 
the first base of the first codon that is expressed by the sequence being 
discussed, and the amino acid numbers are consecutively numbered with the 
first being the amino acid expressed by bases 1-3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
In accordance with the invention, expression vectors suitable for 
prokaryotic systems and expression vectors suitable for eukaryotic systems 
are prepared. DNA encoding amidating enzyme useful in these vectors may be 
isolated as taught in the parent U.S. patent application hereto, (Ser. No. 
086,161 filed Aug. 14, 1987 priority of which has been claimed and the 
entire disclosure of which has been incorporated herein by reference). 
Alpha-amidating enzymes have been isolated from rat fron a rat cell line, 
and purified to homogeneity as taught in the above-identified parent 
application and in grandparent application Ser. No. 655,366 filed Sep. 27, 
1984, now issued as U.S. Pat. No. 4,708,934, priority of which has been 
claimed and the entire disclosure of which has been incorporated herein by 
reference. Amino acid sequences have been determined for the purified 
alpha-amidating enzyme, and these sequences have been used to project a 
variety of oligonucleoteide probes which have been radiolabelled and 
utilized for isolating cDNAs for amidating enzyme. 
The isolated cDNAs have been used to screen libraries prepared, for 
example, from the total RNA of rat medullary thyroid carcinoma tissues, 
their derived cell lines or frorii cell lines known to produce amidating 
enzyme, for example, biological deposit ATCC 75168 (in Vitro 
International, Linthicum, Md.) (Rat MTC tissue) or derived cell line ATCC 
CRL 10919. Total RNA was prepared and Poly-A RNA was selected with oligo 
DT cellulose. cDNAs were prepared by well known methods utilizing first 
reverse transcriptase and then a DNA polymerase. The cDNA was used to 
generate cDNA libraries in the vector .lambda.gt 11 and the recombinant 
DNA's were packaged in vitro to form infectious bacteriophage particles. 
Extracts for packaging are commercially available for examrple from Promega 
Biotech or Clontech Laboratories or can be prepared according to methods 
well-known in the art. The phage were screened with radlolabelled 
oligonucl eotide probes prepared as set forth above. Screening for 
bacteriophage containing alpha aitidati.ng enzyme cDNA ("AE cDNA") was 
accomplished by plating samples of bacteriophage and lifting the phage 
onto nitrocellulose filter discs. Hybridization with two or more 
radiolabelled AE-specific oligonucleotide probes conferred specifilcilty. 
Oligonucleotide probes denoted AE4, AE5, AE8 and AE9 at paces 61-64 of 
parent U.S. application Ser. No. 086,161 (filed Aug. 14, 1987) are 
especially preferred when screening libraries prepared from biological 
deposit ATCC CAL 10919 identified above. 
Analysis o f the AE cDNAs from many bacteriophage isolated by the above 
oligonucleotide hybridization screening procedures indicated that the 
cDNAs could be separated into a plurality of distinct types. The structure 
of one type is shown in FIGS. 5A-5F ("Diagram A" or "Type A sequence") 
where the nucleotides have been numbered with base 1as the first base of 
the codon for the initiator methionine. Below the nucleotide sequence is 
given the single letter amino acid code for translation of the gene 
seauence into protein. Numbers in parentheses at the end of lines indicate 
amino acid numbers. 
Another type of CDNA sequence isolated by the oligonucleotide hybridization 
screening set forth above is represented by FIGS. 6A-6F ("Diagram B" or 
"Type B sequence") where the numbering and other conventions are the same 
as those stated for FIGS. 5A-5F 
Applicants have utilized cDNAs isolated in the above manner for 
constructing both prokaryotic and eukaryotic expression vectors and a 
number of hosts have been transfected with these vectors for effective 
expression of alpha-amidating enzyme. 
In preferred embodiments of the invention, applicants have made novel 
modifications to the foregoing cDNAs in order to optimize not only 
expression, but also recovery of amidating enzyme. The nature and extent 
of modification may vary with the host and/or vector selected. For 
example, applicants insert, in preferred embodiments, a stop codon 
upstream from a sequence which would otherwise code for a membrane 
spanning domain. The presence of these membrane spanning domains may be 
undesirable for a recombinant DNA expression system since they may cause 
the expressed protein to be membrane associated and possibly inactive in 
the host organism or cell line. Examples of membrane spanning domains 
appear in the two cDNA examples set forth in diagrams A and B above (about 
bases 2275-2355 of the sequence shown in diagram A and about bases 
2587-2667 of the sequence shown in diagram B). The cDNAs of diagrams A and 
B are substantially identical on the amino side of these transmembrane 
domains with the exception of what appears to be an intron region from 
base 1178 through base 1492 of the type B cDNA. 
The cDNAs of diagrams A and B above encode protein products of 
approximately 94 and 105 kD, respectively. Both of these proteins are 
larger than mature, active enzymes that have been purified from animal 
tissue extracts or cell line secretions. Each of these primary translation 
products are pre-proerzymes that contain membrane-spanning domains in the 
C-terminal one-third of the ccding sequence. It is preferred that the stop 
codon be placed so that the expressed protein has a molecular weight of 
about 75 kd for when expressed by the cDNA of diagram A (i.e. the stop is 
placed between bases 2025 and 2275) and 87 kD when expressed by the cDNA 
of diagram B(i.e., the stop is placed between about bases 2340 and 2690). 
For cytoplasmic expression of the mature alpha ainidating enzyme in E. 
coli, for example, it is preferred that the gene sequences that encode the 
natural secretory signal sequence be removed, that an initiation codon be 
placed within about 50 nucleotides of the gene sequences encoding the 
start of the mature protein corresponding to alpha-amidatinq enzyme, and 
that the gene sequences encoding the membrane spanning domain in the 
C-terminal region not be translated. The initiating codon is of course 
in-frame with the sequence which encodes the enzyme and, in some preferred 
embodiments, is upstream from the region, sometimes immediately upstream. 
When the AE cDNA was expressed in E. coli, it was discovered that the 
natural gene sequence contained a cryptic E. coli ribosome binding site 
("RBS") and initiation codon internal to the natural initiation sequences. 
This resulted in the production of an N-terminally truncated amidating 
enzyme protein. While this did not prevent the production of the desired 
product in E. coli, the coexistence of the correctly initiated and 
internally initiated products complicates the processing and purification 
of the recombinant product to a useful form and is therefore undesirable. 
To eliminate the unexpected, undesired product, it was necessary to 
eliminate either the ribosome binding site, the internal initiation codon 
or both of these. 
For example, in certain preferred embodiments of the invention, a valine 
codon which, In prokaryotic systems, codes an initiating methionine, is 
altered by a point mutation to an equivalent non-initating valine codon at 
bases 661-663 (of the cDNAs of either diagram A or B). In lieu of this 
point mutation or in addition thereto, applicants, in other preferred 
embodiments, delete or substantially modify any region coding for a 
ribosome binding site whicrh occurs just upstream of an internal 
initiation site, and more preferably any internal ribosome binding site 
whenever one may occur. These modifications are made to substantially 
eliminate internal initiation such that the protein expressed because of 
internal initiation is not observed as a separate band following 
electrophoresis. 
To obtain expression of secreted, active alpha amidating enzyme protein, 
from a recombinant eukaryotic host cell line it was necessary to remove 
the gene sequences encoding the transmembrane domain found in the 
C-terminal region of the natural gene sequences. For the type A CDNA this 
has been done by truncation of the protein coding region through 
introduction of a stop codon at or near to where the natural amidating 
enzyme is post-translationally processed in some natural systems as 
explained in detail below. For the type B CDNA this has also been done by 
introducing a new stop codon in the region of the enzyme protein where the 
natural type B amidating enzyme is post-translationally processed (see 
below). This should not be taken to exclude the possiblity that in some 
host cell systems it may be preferable to express the entire naturally 
occuring gene sequences. Because the type B cDNA contains sequences with 
the characteristics of an unprocessed intron there may be a difficulty in 
expressing this cDNA in some eukaryotic host cells. These cells may not 
efficiently produce an mRNA from the type B gene due to the presence of 
the paired splice donor and acceptor sites. ElimLnation of the acceptor 
site might therefore be necessary to allow for efficient expression of 
type B AE cDNA 
We have discovered that the carboxyl end of the naturally occurring 75 kD 
alpha amidating enzyme protein occurs beyond amino acid position 709 (814 
of type B). To produce the 75 kD protein (87 kD of type B) in a 
recombinant DNA host cell, a stop codon has been introduced into the cDNA 
by mutation of the codon for the lysine of amino acid position 716 (821 of 
type B). This modification has been made using oligonuclectide directed 
site specific mutagenesis. Such mutagenesis can be accomplished in a 
variety of ways. The methods have been reviewed extensively in the 
molecular biology literature. The general method that we have used was 
described by Taylor, J. W. et al. (1985), Nucl. Acids Res., 13: 8749-8764; 
Taylor, J. W. et al. (1985), Nucl. Acids Res., 13: 8764-8785; Nakamaye, K. 
and Eckstein, F. (1986), Nucl. Acids Res., 14: 9679-9698. The reagents 
needed to practice theis method are available in the form of a mutagenesis 
kit from Amersham Corporation. 
The mutation of the sequence that we have produced changes the AAA lysine 
codon to a TAA stop codon. The oligonucleotide used for the mutagenesis 
incorporated this change but was otherwise identical in sequence to the 
naturally occurring cDNA sequence for the respective enzyme (type A or 
type B) being mutated. 
We have also discovered that a naturally occurring shortened torm of the 
alpha amidating enzyme protein is produced by processing of the type B 
protein at the internal region of the protein that is unique to the type B 
enzyme protein. This results in an enzyme product that is approximately 43 
kD in molecular masse Without intending to be bound by theory, it is 
believed that the DNA sequence upstream from the intron region is 
sufficient to code for a polypeptide capable of exhibiting significant 
alpha-amidating activity. Accordingly polypeptides which are easy to 
recover and which are capable of expressing alpha-amidatina activity may 
be encoded by cDNAs which are significantly truncated by placement of a 
stop codon somewhere in the intron region of type B cDNA in just before or 
after the corresponding location where this intron is missing from TYPE A 
cDNA. Preferred truncation results from placement of a stop codon within 
about 30 bases of the beginning of the of the intron region, preferably 
immediately downstream therefrom. To enable the production of one 
preferred short form of alpha amidating enzyme protein in recombinant host 
cells, a modified cDNA is created having a stop codon in place of the 
lysine codon at amino acid position 436 of the type B cDNA. This mutation 
was accomplished by oligonucleotide directed site specific mutagenesis of 
the type B AE cDNA. 
While the shortening of the amidating enzyme protein by introduction of the 
stop codon at amino acid position 436 of the type B cDNA gives a protein 
that most closely approximates the one produced naturally by proteolytic 
cleavage of the primary translation product (or some other cleavage 
intermediates in the biosynthetic pathway), a further shortening of the 
amidating enzyme protein may also result in production of an active 
product in recombinant DNA host cells. We have modified the AE cDNA in 
several other ways to create such shorter forms of protein. In one 
example, we have used oligonucleotide directed site-specific mutagenesis 
to convert a tyrosine codon at amino acid position 396 of the type B cDNA 
to a stop codon. This change will result in a protein that is 
approximately 39 kD when the cDNA is translated and processed. In a second 
case, we have utilized the naturally occuring Bam Hl enzyme recognition 
site of the type B cDNA to introduce a stop codon by linker mutagenesis. 
This method is well known in molecular biology and simply involves the 
cleavage of the cDNA followed by ligation to a double stranded synthetic 
linker fragment that is complimentary to one end of the cleaved cDNA and 
that introduces an in frame stop codon just beyond the cleavage site. We 
have used an oligonucleotide fragment with the following sequence to 
accomplish this modification: 
.sup.5' GATCCACTAATGATCA.sup.3' 
.sub.3' GTGATTACTAGTTCGA.sub.5' 
This linker introduces a stop codon following the histidine codon at amino 
acid 469. Translation and processing of the cDNA once it has been modified 
in this fashion results in the synthesis of a protein of approximately 46 
kD. 
Preferred placement of a truncating stop codon is within about 15 bases of 
a DNA sequence which codes for consecutive basic residues (usually a 
Lys--Lys) and especially immediately upstream therefrom. Without intending 
to be bound by theory, it is believed that the natural polypeptide coded 
by the cDNAs of type B is processed, during post-translational 
modifications which occur during natural expression of amidatlng enzyme, 
at or near such consecutive basic residues, for example, the consecutive 
lysines coded within the intrcn region of the cDNA of diagram B. Even when 
the inserted stop codon-s are not intended to truncate the expressed 
polypeptide in the above-described manner, it is preferred that the 
inserted stop codon be placed within about 20 bases, and preferably 
immediately upstream from, DNA sequences coding for consecutive basic 
amino acid residues. insertion of stop codons at these positions will 
likely result in expression of a polypeptide resembling certain natural 
amidating enzymes after they have undergone post-translational processing. 
For cytoplasmic expression in prokaryotic systems, any signal sequence 
coding regions (for example, the first bases of both the type A and type B 
cDNAs diagrammed previously) are preferably eliminated and a methionine 
initiator codon is inserted within about 50 nucleotides of the beginning 
of the region which codes for amidating enzymes. 
An alternative embodiment for prokaryotic expression eliminates any coding 
sequences for signal sequence or proenzyme sequence and inserts an 
initiator methionine codon within about 50 nucleotides of the beginning of 
the region which codes for amidating enzyme. In many natural AE cDNAs, 
this corresponds to the beginning of the region which encodes ser-x-ser (X 
being phe or leu). See, for example, bases 124 to 132 of the sequence for 
type A or type B cDNA. In some embodiments secretion of alpha amidating 
enzyme may be desirable. in this case it is preferable to retain the 
signal sequence coding regions, or alternatively to replace them with 
heterologous sequences that can serve the same function, for example, the 
signal sequences of the bacterial OMP A protein. 
It will be readily apparent to those skilled in the art that numerous 
mutations and truncations of the DNA sequences set forth herein for 
encoding amidating enzyme are possible within the scope of the invention 
and that such modified sequences would code for polypeptides capable of 
functioning as amidating enzymes. Accordingly, applicants claims should be 
construed to include all functional equivalents of DNA sequences, 
expression vectors and host cells specifically set forth. 
Examples of prokaryotic expression vectors which may desirably be modified 
to include DNA sequences encoding amidating enzyme in accordance with the 
invention include but are not limited to pKK233-,pKK322-2, pPROK-1, 
pkT279,280,287, pPL lambda, pYEJ001, pKC30, pPROK-C, all commercially 
available. Prokaryotic hosts which may be transfected with expression 
vectors in accordance with the invention include but are not limited to 
C600, LE392, RR1, DH1, SF8, all commerically available. 
Eukaryotic expression vectors which may desirably be modified to include 
DNA sequences encoding amidating enzyme in accordance with the invention 
include but are not limited to pMAMNeo, pdBPVMMTNeo, pRSV, peuK-C1, 
pCH110, all commerically available. Appropriate yeast vectors may also be 
used. Preferred eurokaryotic hosts may be transfected with expression 
vectors in accordance with the invention include but are not limited to 
ATCC deposit CRL 10919, Hela, CV1, C127, CHO (Chinese Hamster Ovary) and 
COS. 
EXAMPLE 1 
Expression of Alpha Amidating Enzyme Proteins in E. coli 
In order to express alpha amidating enzyme in E. coli (see the flow chart 
of FIG. 2), a cDNA fragment having the sequence set forth in diagram A, 
above, was digested with KpnI and Hind III and the fragment of about 2.1 
kb was isolated. To build back a amino terminus corresponding to a natural 
mature enzyme, an oligonucleotide linker with the sequence 
.sup.5' CATGTCATTTTCCAATGAATGCCTTGGTAC.sup.3' 
.sub.3' AGTAAAAGGTTACTTACGGAAC.sub.5' 
was ligated to this DNA fragment. The resulting fragment contained one Nco 
I compatible sticky-end and one Hind III sticky end. The E. coli 
expression vector pK233-2was obtained commercially from Pharmacia and 
digested with restriction enzymes Nco I and Hind III. The large linear 
fragment was isolated and ligated to the linker adapted cDNA fragment. The 
ligation mixture was used to transform competent E. coli JM105. 
Transformants were selected by ampicillin resistance and the clones 
isolated were analyzed for the recombinant plasmid by restriction enzyme 
and DNA sequence analysis to confirm the structure of the expression 
vector (hereafter "pAE12") that they contained. The expression vector 
contains the hybrid trp-lac promoter which is repressed by the lac 
repressor and inducible by treatment of the cells with 
isopropylthiogalactoside (IPTG). Upstream from the initiator methionine 
tlhe vector also contains the sequences of a strong ribosome binding site. 
To obtain expression of the alpha amidating enzyme in the E. coli, the 
recombinant cells were grown with shaking in LB-broth at 37.degree. C. to 
an CD.sub.600 of 0.4. IPTG was added to the culture to a final 
concentration of lmM and the growth was allowed to continue at 37.degree. 
C. with shaking for three to five hours. Cells were collected by 
centrifugation of the culture and the supernatant was discarded. The cells 
were resuspended in buffer containing a coctail of protease inhibitors, 
treated with lysozyme and then sonicated to lyse the cell membranes. The 
lysates were centrifuged at 12,000.times.g to separate the soluble and 
insoluble fractions of the cells. Each fraction was analyzed by SDS-PAGE 
and protein staining. The alpha amidating enzyme protein was readily 
identified as an IPTG inducible product in the insoluble protein fraction. 
Since the initial expression plasmid did not contain a stop codon 
specified by the alpha amidating enzyme gene sequences, the inducible 
product formed contains sequences specified by downstream vector DNA fused 
to the C-terminal of the alpha amidating enzyme protein sequences. In 
addition, the induced insoluble protein also contained a smaller amidation 
enzyme specific protein that represented a product formed by internal 
initiation of protein synthesis at a cryptic RBS and initiation codon 
(amino acid position 221 of the alpha amidating enzyme sequence). 
To remove the unwanted sequences from the C-terminal portion of the 
expressed product, a mutation of the lysine codon at position 716 of the 
type A sequence was made to generate a TAA stop codon at this position. 
The mutated cDNA was then digested with Kpn I and Eco R1 and used to 
replace the original Kpn I-Eco R1 fragment in the initial expression 
vector pAE12. In a similar fashion, the type B cDNA sequences were mutated 
at the comparable position (amino acid 821) to create a stop codon and the 
Kpn I-Eco R1 fragment from the mutated type B cDNA was used to replace the 
corresponding fragment in pAE12. The two expression plasmids so created 
pAE24 (type A) and pAE25 (Type B) were then used to transform JM105. The 
resulting strains were cultured for expression as was done previously for 
pAE12-containing strains. The pAE24 was found to produce two IPTG 
inducible, insoluble proteins of approximately 75 kD and 55 kD while the 
pAE25 was found to produce two IPTG inducible insoluble proteins of about 
87 kD and 67 kD. Again, the small protein in each of these pairs 
represents the unwanted amino-terminally truncated product from either the 
type A or type B cDNA. 
To eliminate the initiation of protein synthesis at the cryptic internal 
ribosome binding site and initiation codon (amino acid position 221) the 
GTG start codon, (GTG can serve as an initiator met codon in bacteria), 
was converted to a GTT codon that cannot initiate protein synthesis but 
which still encodes the valine that is normally found at this position in 
alpha amidating enzyme proteins encoded by natural genes. When the mutated 
region of the cDNA was substituted for the natural sequence in the 
expression vectors pAE24 and pAE25, two new vectors were created, pAE31 
and pAE32. Transforming E. coli JM105 with these modified expression 
vectors and testing protein production from the resulting recombinant 
strains indicated that this mutagenesis was effective in eliminating the 
unwanted internal initiation. The IPTG induced product from the host cells 
carrying pAE31 was found to be 75 kD while that from cells transformed 
with pAE 32 was found to be 87 kD. 
Since we have found that naturally occurring amidating enzyme from type B 
cDNA is post-translationally processed to give proteins of approximately 
43 kD, we have prepared a series of mutations in type B AE cDNA that 
allows expression of proteins that terminate at or near the position where 
the naturally processed enzyme ends. Two of these mutations were prepared 
by oligonucleotide mutagenesis while a third was created by adapter-linker 
mutation as indicated above. When cDNAs carrying these mutations were used 
to replace the corresponding segments of pAE32, transformed into JM105 and 
analyzed for protein production in experiments similar to those described 
above, truncated alpha amidating enzyme proteins were detected. With a 
mutation at amino acid position 396 of type B cDNA changing a natural 
tyrosine codon to a stop codon (pAE36), a 39 kD enzyme protein was found 
while a linker mutagenesis that ended translation at the histidine codon 
of amino acid 464 resulted in a vector, pAE51, which produced a 
recombinant alpha amidating enzyme protein of 46 kD following 
transformation and induction of E. coli JM105. 
All of recombinant alpha amidating enzyme proteins produced in E. coli 
described above were found to segregate with the insoluble fraction of the 
cell extracts. The enzymes could be rendered soluble and active by 
treatment with 8M urea followed by rapid dilution in 50 mM Tris-HCl pH7. 
When E. coli JM105 carrying pAE12 was grown and induced with IPTG as 
described, the alpha amidating enzyme proteins were present at levels of 
at least 30 mgs per liter of bacterial culture. 
Representative samples of the induced insoluble protein produced in E. coli 
carrying AE expression plasmids are shown in FIGS. 3 and 4. 
EXAMPLE 2 
Generation of mammalian expression vector ud BPV-MMTNEO-AE.sub.A75 
To generate a mammalian expression vector which expresses and 
constitutively secretes 75 kD type A alpha amidating enzyme from mammalian 
cells (see the flow chart of FIG. 1), the following was performed: 
1) The intermediate expression vector pdMMTNeo (commercially available from 
American Type Culture Collection) (as shown) was digested with Bgl II. The 
linear form was isolated and purified. 
2) The recombinant type A cDNA containing the full prepro sequence and an 
artificial stop codon TAA at position 2146-2148 was isolated by sequential 
digestion with Bgl I and Xho I. The fragment corresponding to alpha 
amidating enzyme was then isolated and purified. 
3) The insert (type A alpha arnidating enzyme) and vector (pdMMTNeo) were 
mixed and the corresponding ends were made flush using the Klenow fragment 
of DNA polymerase I. The 5' protruding segments were filled in with added 
dNTP, and the 3' protruding segments were digested back to produce a flush 
end (alternatively sequential Sl nuclease and Klenow+dNTP could be 
utilized for producing flush ends). The flush ended molecules were then 
ligated for 16 hours at 15.degree. C. 
4) The ligated material was then transformed into E. coli RRI. Recombinant 
clones were selected in the presence of 50 ug/ml ampicillin. The 
orientation of the insert in the recombinant clones was verified using a 
battery of restriction enzymes. One clone which was referred to as 
pdMMTNeo .varies.-AE.sub.A75 (clone 11) was determined to have the type A 
cDNA in the correct orientation with respect to the MMT promoter. 
5) Plasmid DNA from recombinant pdMMTNeo .varies.-AE.sub.A75 (clone 11) was 
digested with BamHI. The linearized vector was isolated and purified and 
then treated with bacterial alkaline phosphatase (B.A.P.) for 2 hours at 
37.degree. C. to remove 5' phosphates. The BPV-1 genome was isolated and 
purified following B and BamHI digestion of the vector pdBPVMMTNeo. This 
BamHI fragment of BPV-1 DNA, which is approximately 8.0 kb, was then 
ligated to the BamHI linearized and B.A.P. treated pdMMTNeo 
.varies.AE.sub.A75 vector, for 3 hours at 14.degree. C. After the ligation 
mixture was transformed into E. coli RR1, the recombinant clones were 
selected on 50 ug/ml Ampicillin LB agar plates. The recombinant plasmids 
were analyzed for BPV DNA and were also analyzed for type A AE cDNA. 
Restriction mapping revealed that clone 21 was approximately 17 kb and 
produced a restriction map as expected. This expression plasmid was then 
used for expression of .varies.AE.sub.A75 in mouse C127 cells. 
6) Mouse C127 cells were transfected with 20 ug of pdBPV-MMTNeo 
.varies.AE.sub.A75 by the standard CaPO.sub.4 precipitation technique. 
Approximately 2 weeks post transfection, transformed foci were 
individually picked and grown in growth media containing the antibiotic 
G418. When cells were grown to a sufficient capacity in Dulbecco's 
Modified Eagle Medium plus 10% fetal calf serum, the clones were assessed 
for the ability to secrete Alpha Amidating Enzyme by measuring the 
enzymatic activity in the conditioned cell culture media, as well as by 
measuring the alpha amidating enzyme immunoreactivity in the medium using 
standard radiolabelling and immunoprecipitation techniques. Clones 
secreting active, immunoreactive 75 kD alpha amidating enzyme were 
expanded to large numbers of cells (switched to cell culture medium with 
reduced serum and therefore reduced level of exogenous protein) and are in 
use to produce large quantities of active recombinant enzyme from the cell 
conditioned media. 
The terms and descriptions used herein are embodiments set forth by way of 
illustration only, and are not intended as limitations on the many 
variations which those of skill in the art will recognize to be possible 
when practicing the present invention, as defined by the following claims.