Enzymatic process for producing a desired protein from an amino terminal extended protein

An enzymatic process is presented for producing a desired protein from an amino terminal extended protein by reaction with one or more aminopeptidases, glutamine cyclotransferase and pyroglutamine aminopeptidase.

The invention relates to an enzymatic process for producing a desired 
protein from an amino terminal extended protein. 
Over the past decade, the principle of a new protein purification technique 
has emerged as a result of recombinant DNA technology. DNA, encoding 
additional polypeptide or protein tags, is fused to the gene of interest. 
Expression of these gene fusions results in protein fusions which may be 
identified, analyzed and purified by techniques using the properties of 
the additional polypeptide tag. This has in certain cases eliminated the 
need for extensive screening and optimization procedures previously 
required for purification. 
Polypeptide tags specifying a variety of different biospecific and 
biochemical interactions can be or have been utilized as the basis for 
fusion tag techniques. These include (1) entire enzymes with affinity for 
substrates or inhibitors; (2) peptide-binding proteins; (3) 
carbohydrate-binding proteins or domains; (4) biotin-binding domains; (5) 
antigenic epitopes with affinity for specific immobilized monoclonal 
antibodies; (6) charged amino acids for use in charge-based recovery 
methods; (7) polyhistidine residues for binding to metal chelates and 
subsequent recovery by immobilized metal affinity chromatography and (8) 
other polyamino acids with various binding specificities. 
Provided that the added polypeptide tag can be effectively and specifically 
removed, this technology should find application in areas from basic 
research to industrial production. On a laboratory scale, the fusion tag 
technique should be a powerful and elegant tool for one-step recovery and 
purification of a large number of authentic recombinant proteins. On an 
industrial scale, the technology could be used in the recovery and 
purification of high-cost pharmaceuticals and other proteins prepared by 
using recombinant technology or other technology. 
Unfortunately, the use of this protein purification principle has been very 
limited because of a major and serious drawback. It is often difficult, 
impossible or too expensive to remove the additional terminal sequence 
from the desired protein product. This is most often necessary to do in 
order to obtain the desired correct protein function, structure and other 
properties. 
This is of the utmost importance when dealing with basic research covering 
e.g. function studies of proteins (enzymes, receptors, hormones, etc.). 
It is also of the utmost importance when dealing with proteins for 
pharmaceutical use in which case it is essential to avoid undesired 
immunological and other response effects of the pharmaceutically active 
protein which therefore has to have the correct and natural structure, 
i.e. without any added polypeptide or single amino acid. 
One solution to the problem has been to remove the added polypeptide tag by 
using relatively drastic conditions and chemical cleavage reagent, such as 
CNBr in 70% formic acid, or hydroxylamine treatment at basic pH. The 
methods have several major drawbacks which include the use of 
protein-destroying conditions and very toxic chemicals, this leading to 
high levels of unwanted degradation of the protein of interest and serious 
health problems for the personnel involved in the use of these techniques. 
Another approach is to use so-called specific endoproteases. The blood 
clotting factor Xa has a proteolytic specificity for the tetra peptide 
sequence IleGluGlyArg and has been used to liberate different added 
sequences from different proteins. A sequence allowing a relatively 
specific cleavage by collagenase has also been proposed. Another sequence 
of five amino acids can be cleaved by the enzyme enterokinase. 
Another example using this technique is known from DE 29 22 496 which 
partly corresponds to U.S. Pat. No. 4,543,329. This document discloses the 
preparation of proteins from C-terminal extended proteins. The protein is 
extended with 4 amino acids, where the extension is Pro-Xyz-Gly-Pro, and 
Xyz is any amino acid. The tetrapeptide is removed using the enzymes 
collagenase, an aminopeptidase and a proline aminopeptidase. No stop point 
is used for collagenase. Purification after removal is difficult, see the 
following. Further, proline is not the optimal amino acid as terminal, if 
it is desired to incorporate specific characteristics in the extension, 
because it reduces the movability of the extension. This means that it is 
more difficult to design an extension for a given purpose. 
The above-mentioned types of endoproteases have thus in principle the 
ability to cleave at the C-terminal of their recognition sequence, 
resulting in liberation of the desired protein with the correct amino 
terminal. 
The use of enzymatic methods using sequence specific endoproteases has, 
however, several major drawbacks. One of these is that the desired product 
should not contain the recognition sequence for the endoprotease used, 
because this will result in undesired degradation of the product. 
Furthermore, the selectivity is not always as precise as anticipated, 
because the enzyme can cleave at more or less homologous sequences in the 
desired product, or because the enzyme contains contaminating homologous 
endoproteases (which therefore are difficult to remove or inactivate), all 
of which will result in undesired modification or degradation of the 
protein product. 
A further problem in connection with the use of sequence specific 
endoproteases is that the potentially useful enzymes are very often 
(extremely) expensive in use, especially when producing proteins for 
pharmaceutical use. 
To overcome the problems associated with the chemical and endoproteolytic 
methods described above, it is in certain cases possible to use 
exoproteases to remove the amino(N)- or carboxy(C)-terminal extensions 
(amino- and carboxy-peptidases, respectively). 
These enzymes remove an amino acid or a dipeptide from the unblocked N- or 
C-terminal of a peptide/protein. In order to fully control the removal of 
a terminal extension there must be a stop point at the N- or C-terminal, 
respectively, of the desired protein. If no such stop point exists, the 
result will be an unwanted further degradation of the protein. 
An example of the use of an exopeptidase in connection with the removal of 
a terminal extension from a protein with a natural stop point is the 
use-of the enzyme dipeptidyl aminopeptidase I (DAP I; EC 3.4.14.1), see EP 
217 814 and DK 166 687. DAP I is used in both cases to produce a desired 
protein containing an internal stop point within the protein itself. 
DAP I is an enzyme which removes two amino acids, as a unit, from the 
N-terminal of a protein or polypeptide. Under appropriate conditions, 
dipeptide removal will commence and continue unless or until (1) the amino 
group of the N-terminal is blocked, (2) the site of removal is on either 
side of a proline, or (3) the N-terminal amino acid is lysine or arginine. 
DAP I can thus be used to remove N-terminal extensions from a protein 
containing a proline as the second or third amino acid, or N-terminal 
lysine or arginine residue. 
Another example of the use of an exopeptidase in connection with the 
removal of a terminal extension from a protein with a natural stop point 
is the use of the enzyme aeromonas aminopeptidase (AAP), which removes 
single amino acids from the N-terminal of a protein or polypeptide, cf. EP 
489 711 and EP 191 827 disclosing removal of N-terminal residues by means 
of an aminopeptidase, e.g. AAP. Under appropriate conditions, amino acid 
removal will commence and continue unless or until (1) the amino group of 
the N-terminal is blocked, (2) the site of removal is on the N-terminal 
side of proline, or (3) the N-terminal amino acid is glutamic acid or 
aspartic acid. 
Danish patent application no. 3245/89 corresponding to EP 348 780 discloses 
a process for preparing a protein from N-terminal extended protein, which 
is extended with at least a proline residue just in front of the protein. 
It is suggested that the process is useful when the extension contains 
more than one amino acid residue in front of the proline residue, but this 
is not possible. The enzyme used Aminopeptidase P, can only remove the 
amino acid residue just in front of the proline residue (Yoshimoto, T. et 
al., J. Biochem., Vol. 104, p. 93-97 (1988)). 
This means that the method is only useful for removing N-terminal extension 
having only two amino acid residues and having the sequence Xaa-Pro (where 
Xaa is any amino acid residue). 
DAP I and AAP may thus have some usefulness in the production of a desired 
recombinant protein from a precursor protein by constructing such 
precursor protein to contain a removable N-terminal extension, i.e. an 
extension which does not contain any of the above-mentioned stop points. 
Treatment of the precursor protein with either of the two enzymes may 
result in removal of the N-terminal extension. 
This process, however, may be severely limited in its application, as 
dipeptide or amino acid removal by DAP I or AAP, respectively, will 
continue sequentially and unhindered until one of the aforedescribed 
termination sequences (stop point) is reached. Thus, the aminopeptidase 
approach should find limited use, being applicable generally only in those 
instances in which the N-terminal portion of the desired protein product 
is itself a DAP I or AAP stop point. 
It is also known to remove N-terminal methionine from angiogenine by means 
of AAP followed by non-enzymatic cyclization, cf. Analytical Biochemistry 
175, 450-461 (1988). 
It has now surprisingly been discovered, however, that it is possible to 
design a generally removable N-terminal extension containing a removable 
stop point at the C-terminal of the extension, i.e. immediately before the 
N-terminal of the desired protein product, thus avoiding the need for an 
internal stop point in the desired protein product. 
SUMMARY OF THE INVENTION 
The present invention provides enzymatic processes for producing a desired 
protein from an aminoterminally extended version of the protein. 
Typically, the starting proteins have the formula 
EQU NH2-A-Glutamine-Protein-COOH, 
wherein A represents one or more amino acids aminoterminal to a glutamine 
residue and protein represents the desired protein product. In practicing 
the invention, the aminoterminally extended protein is contacted either 
simultaneously or sequentially with: (a) one or more aminopeptidases; (b) 
glutamine cyclotransferase (GCT); and (c) pyroglutamine aminopeptidase 
(PGAP). The first aminopeptidase(s) catalyze(s) the removal of the amino 
acids aminoterminal to the glutamine; glutamine cyclotransferase then 
catalyzes the conversion of the glutamine to pyroglutamine; and, finally, 
pyroglutamine aminopeptidase catalyzes the removal of the pyroglutamine to 
produce the desired protein product. 
Aminopeptidases that may be used in practicing the invention include 
without limitation dipeptidyl aminopeptidase I (DAP I), aeromonas 
aminopeptidase (AAP), aminopeptidase P (APP), and proline iminopeptidase 
(PIP). The aminoterminally extended starting protein may be reacted with 
the enzymes sequentially or simultaneously. The only requirement is that 
the reaction conditions used support the enzymatic activity of each 
enzyme. Schematically, a protein with such a removable N-terminal 
extension can be written as 
EQU A-Glutamine-Protein I 
wherein Protein is the desired protein, Glutamine is a glutamine residue 
attached directly to the amino terminal of the desired protein, and A is 
an amino acid sequence which is attached directly to the glutamine 
residue. 
It has surprisingly been found that the desired protein can be obtained by 
using the process according to the invention which is defined in the 
characterizing portion of claim 1. 
The term aminopeptidase, except for pyroglutamine aminopeptidase, as used 
in the specification covers usual aminopeptidases acting on amino acids 
with primary or secondary amino groups. 
The protein I is preferably first reacted with one or more aminopeptidases 
and simultaneously with glutamine cyclotransferase and then with 
pyroglutamine aminopeptidase. 
In some cases it is also possible to react the protein I with all enzymes 
simultaneously, e.g. in a mixture. 
Further, it is in some cases possible first to react protein I with one or 
more aminopeptidases and then simultaneously with glutamine 
cyclotransferase and pyroglutamine aminopeptidase. 
It is also possible to react protein I with one enzyme at a time. 
It is preferred to use dipeptidyl aminopeptidase as aminopeptidase. 
Good results are also obtained by using aeromonas aminopeptidase as 
aminopeptidase. 
It is moreover possible to use more than one aminopeptidase, e.g. 
dipeptidyl aminopeptidase and aminopeptidase P or aeromonas aminopeptidase 
and aminopeptidase P. 
The choice of aminopeptidase(s) depends on the amino acid sequence in A or 
vice versa. A is i.a. constructed according to the used aminopeptidase(s). 
The aminopeptidases are chosen according to their suitablity in connection 
with the specifically constructed extended protein. 
The A-sequence can e.g. be an amino acid sequence designed to be removed by 
DAP I in the presence of the enzyme glutamine cyclotransferase (GCT), and 
it must therefore have an even number of amino acids where the first amino 
acid is different from lysine and arginine, all other uneven amino acids 
are different from proline, glutamine, lysine and arginine, and all even 
amino acids are different from proline. 
A desired protein with glutamine as the only N-terminal extension left can 
be cyclizised to the pyroglutamine by the enzyme (GCT), i.e. it is 
converted to an amino acid with a blocked amino group, and aminopeptidases 
cannot proceed through this amino acid residue. 
Then, by using one or more aminopeptidases together with a surplus of GCT 
in an enzymatic reaction containing the N-terminal extended protein 
product, GCT will cyclizise the glutamine residue to a pyroglutamine 
residue, as soon as the aminopeptidases, such as DAP I or AAP, have 
removed all the amino acids before the glutamine residue. This cyclization 
reaction will thus result in a blocked N-terminal immediately before the 
desired protein product, and the reaction catalyzed by the 
aminopeptidase(s) will not proceed any further. 
If the desired protein has the amino terminal sequence Xaa-Pro-, there is 
no need for a simultaneous reaction with DAP I and GCT, because the DAP I 
reaction will always stop immediately before the glutamine residue due to 
the proline residue in the desired protein. 
This means that the DAP I treatment can be performed alone, and GCT can 
then be added to the reaction mixture. Furthermore, there is no need for 
using a surplus of GCT. 
For the same reason as above, it is possible to perform all three enzymatic 
reactions at the same time by using both DAP I, GCT and PGAP in the same 
reaction mixture. 
The resulting N-terminal pyroglutamine residue can be effectively and 
selectively removed by the enzyme pyroglutamine aminopeptidase (PGAP; EC. 
3.4.11.8), resulting in the desired protein product without any N-terminal 
extension and with the correct and desired N-terminal amino acid. 
Since no enzyme reactions can be expected to lead to 100% conversion, the 
PGAP catalyzed removal of the pyroglutamine residue is very advantageous. 
This is because a protein with blocked amino terminal, i.e. pyroglutamine, 
has a neutral amino acid at the amino terminal, and it will therefore act 
as a protein with an amino terminal amino acid having a free amino group 
and a negatively charged side chain, i.e. glutamic acid and asparagine. 
Then, as the pyroglutamine removal proceeds, the PGAP catalyzed removal of 
the amino terminal pyroglutamine residue from the desired protein results 
in a change in the total charge. This change then enables analytical and 
preparative separation by ion exchange chromatography and electrophoresis 
of the desired protein from the pyroglutamine extended protein. This is of 
the utmost importance in order to follow the enzymatic conversion and in 
order to isolate the desired protein without any pyroglutamine extension. 
If the A-sequence contains one or more proline residues, the enzymatic 
removal will stop one or two amino acids from the first proline residue. 
However, it has surprisingly been found that if the A-sequence contains 
either a single proline residue or two or more adjacent proline 
residues--or a mixture of either single proline residues and/or two or 
more adjacent proline residues separated by an even number of amino 
acids--and no proline residue as the C-terminal amino acid of the 
A-sequence--and an uneven number of amino acids before the first proline 
residue, the entire A-sequence can be removed in a reaction mixture with a 
combination of GCT, DAP I, and the well-examined enzyme aminopeptidase P 
(APP), which is known to selectively remove an amino acid from the 
N-terminal of an amino acid sequence, if the amino acid is followed by a 
proline residue. Furthermore, if the A-sequence contains either a single 
proline residue or two or more adjacent proline residues, or a mixture of 
either single proline residues and/or two or more adjacent proline 
residues separated by an uneven number of amino acids, and a proline 
residue as the C-terminal amino acid of the A-sequence, and an uneven 
number of amino acids before the first proline residue, the entire 
A-sequence can be removed in a reaction mixture with a combination of GCT, 
DAP I, APP, and the well-examined enzyme proline iminopeptidase (PIP) 
which is known to selectively remove an N-terminal proline residue from an 
amino acid sequence. 
Further, it has surprisingly been found that using AAP together with a 
surplus of GCT in an enzymatic reaction containing the N-terminal extended 
protein product, GCT will cyclizise the glutamine residue to a 
pyroglutamine residue, as soon as AAP has removed all the amino acids 
before the glutamine residue. This cyclization reaction will thus result 
in a blocked N-terminal immediately before the desired protein product, 
and the reaction catalyzed by the aminopeptidase(s) will not proceed any 
further. 
By using exopeptidases as described in this invention, undesired and 
unspecific internal cleavage of the desired protein product can be fully 
avoided. 
The enzymes used in the present invention can easily and cheaply be 
produced in a highly purified form.

EXPERIMENTAL DETAILS 
Materials and Methods 
DAP I was prepared from turkey liver essentially according to Metrione, R. 
M. et al. Biochemistry 5, 1597-1604 (1966). The purification procedure 
included the following steps: Extraction autolysis, ammonium sulfate 
precipitation, gel filtration on Sephacryl.RTM. S-300 HR, desalting on 
Sephadex.RTM. G 25 F, and anion exchange chromatography on 
DEAE-Sepharose.RTM. FF. Purified DAP I was stored at -18.degree. C. in 2 
mM sodiumphosphase buffer, 150 mM NaCl 2 mM cysteamine, 50% glycerol, pH 
6.8. 
GCT was prepared from crude papaya latex essentially according to Messer, 
M. and M. Ottesen, Compt. Rend. Trav. Lab. Carlsberg, 35, 1-24 (1965), 
except that CM-Sepharose.RTM. FF replaced CM-Sephadex.RTM. employed by 
Messer and Ottesen. Purified GCT was stored at -18.degree. C. in 4 mM 
sodiumphosphate buffer, 20 mM NaCl, 50% glycerol, pH 7.0. 
PGAP was prepared from extracts of E. coli DH1, harbouring the PGAP 
expression plasmid pBPG 1, described by Yoshimoto, T. et al., J. Biochem. 
113, 67-73 (1993). The purification procedure included the following 
steps: Centrifugation, followed by hydrophobic interaction chromatography 
on phenyl-Sepharose.RTM. FF and buffer exchange on Sephadex.RTM. G 25 F. 
Purified PGAP was stored at -18.degree. C. in 6 mM Tris-Cl, 40 mM NaCl, 2 
mM EDTA, 2 mM cysteamine, 50% glycerol, pH 8.0. 
APP was prepared from extracts of E. Coli DH1, harbouring an APP expression 
plasmid similar to pAPP4, described by Yoshimoto, T. et al, J. Biochem. 
105, 412-416 (1989). The purification procedure included the following 
steps: Centrifugation, ammonium sulphate fractionation, followed by anion 
exchange chromatography of DEAE-Sepharose FF and gel filtration of 
Sephacryl S-300 HR. Purified PGAP was stored at -18.degree. C. in 6 mM 
Tris-Cl, 40 mM NaCl, 0.1 mM Mn.sub.2 Cl, 50% glycerol, pH 7.5. 
The invention is further defined by reference to the following examples. 
EXAMPLE 1 
Removal of His-Ser-Gln from Glucagon 
______________________________________ 
Glucagon 5 mg/ml in 0.01 HCl 
DAP I (20 U/ml) 
100 .mu.l are mixed with 900 .mu.l 100 mM 
dithiothreitol 
GCT (375 U/ml) 
100 .mu.l are mixed with 900 .mu.l H.sub.2 O 
PGAP (20 U/ml) 
100 .mu.l are mixed with 100 .mu.l H.sub.2 O 
Chicken cystatin 
Cl, 50% 1 mg/ml in 10 mM Tris .RTM. 
glycerol, pH 7.5 
______________________________________ 
Step 1 
Removal of His.sup.1 -Ser.sup.2 from glucagon and conversion of Gln.sup.3 
to pGln by DAPI/GCT 
400 .mu.l glucagon (2 mg; 0.57 .mu.mol) with the amino terminal His.sup.1 
-Ser.sup.2 -Gln.sup.3 -Gly.sup.4 -Thr.sup.5 -Phe.sup.6 -Thr.sup.7 
-Ser.sup.8 SEQ ID NO: 1 are mixed with 100 .mu.l DAP I (0.2 unit), 80 
.mu.l GCT (3 units) and 1420 .mu.l 100 mM sodium phosphate buffer, pH 7.0, 
and then incubated at 37.degree. C. After 20 min, 1500 .mu.l are taken out 
in 710 .mu.l H.sub.2 O and 40 .mu.l chicken cystatin (40 .mu.g) (Sigma) 
and incubated for 30 min at 23.degree. C. to inactivate DAP I activity, 
whereafter 1000 .mu.l are desalted on a Sephadex.RTM. G 25 column (NAP 10) 
equilibrated with 10 mM sodium phosphate buffer, pH 7.0 (sample A). 
Step 2 
Removal of pGln from des-His.sup.1 -Ser.sup.2 -Gln.sup.3 pGln.sup.3 
!-glucagon SEQ ID NO: 2 with PGAP 
Another 1200 .mu.l of the chicken cystatin inactivated reaction mixture are 
mixed with 20 .mu.l PGAP (0.2 unit), incubated at 37.degree. C., and after 
5 min 1000 .mu.l are desalted as above (sample B). 
Samples A and B were subjected to amino terminal sequence determination on 
an "Applied Biosystems 477 A Protein Sequencer". No sequence could be 
determined for sample A, which means that it has a blocked amino terminal, 
i.e. pGln. Sample B, however, had the amino terminal sequence 
Gly-Thr-Phe-Thr-Ser, SEQ ID NO: 3 which means that PGAP has removed the 
amino terminal pGln residue. 
Thus, by the sequential treatment with DAP I/GCT and PGAP, the sequence 
His-Ser-Gln has been specifically removed from glucagon. 
EXAMPLE 2 
Production of HisTag2-hTNF.alpha. 
This example describes in details the plasmid construction used to produce 
His-tagged hTNF.alpha. in E. coli. 
Plasmid DNA, BBG18, (British Biotechnology) carrying a modified cDNA 
sequence encoding the mature Human Tumour Necrosis Factor .alpha. 
(hTNF.alpha.) N-terminal extended with methionine is used as template in a 
Polymerase Chain Reaction (PCR) with the use of the synthetic 
oligonucleotides SEQ 5.sup.(1) and SEQ 6 as primers. The amplified DNA 
product is the sequence encoding hTNF.alpha. N-terminal extended with 
glutamine instead of methionine and with a NheI restriction site at the 5' 
end of the glutamine codon. The amplified DNA is subcloned as a NheI-EcoRI 
fragment into the NheI/EcoRI restriction sites of the E. coli expression 
vector, pTrcHis (Invitrogen Corporated) resulting in the plasmid pCLU7. By 
replacement of the small his-tag encoding NcoI-NheI fragment on pCLU7 with 
a synthetic linker (oligonucleotides SEQ 7 and SEQ 8) encoding a modified 
his-tag designed for optimal expression and subsequent removal by the 
process of the invention, the plasmid pCLU14 is obtained. In order to 
bring the hTNF.alpha. gene in frame with the his-tag, pCLU14 is digested 
with NheI followed by a Mung Bean nuclease treatment for removal of single 
stranded extensions to obtain blunt ends. Finally, the DNA is religated to 
give the expression plasmid pCLU15-1. pCLU15-1 encodes hTNF.alpha. 
N-terminally fused to the extension peptide, 
Met-Arg-His-His-His-His-His-His-Gly-Arg-Gln-hTNF.alpha. SEQ ID NO: 4 
FNT .sup.(1) DNA oligonucleotides 
SEQ 5: CTG CAG CTA CCC AGG TCA GAT CAT CTT CGC 
SEQ 6: GGT GAA TTC GGA TCC TTA 
SEQ 7: CAT GCG TCA TCA TCA TCA TCA TCA TGG GCG 
SEQ 8: CTA GCG CCC ATG ATC ATG ATG ATC ATG ACG 
In parallel, other variants of histidine purification tags and other 
purification tags for affinity chromatography are fused to the hTNF.alpha. 
gene by replacement of the NcoI-NheI fragment of pCLU7 by synthetic 
linkers. 
Likewise, the gene encoding the Human Epidermal Growth Factor (hEGF) 
obtained from Invitrogen Corporation on the plasmid BBG7 is modified by 
PCR and incorporated in the pCLU14 plasmid replacing the hTNF.alpha. gene 
to give the expression plasmid pCLU17 encoding hEGF fused to the his-tag. 
N-terminal extended hTNF.alpha. is produced in E. coli strain TOP10 
(Invitrogen Corporated) transformed with expression plasmids. Cells are 
cultured in medium scale (4-8.times.0.5 litre) in SOB +ampicillin at 
37.degree. C. Gene expression is induced by addition of IPTG at OD.sub.600 
=0.6. Cells are harvested by centrifugation after another 4 hours of 
incubation at 37.degree. C. 
EXAMPLE 3 
Generation of authentic TNF.alpha. from 
Met-Arg-6xHis-Gly-Arg-Gln-TNF.alpha. (HisTag2-TNF.alpha. SEQ ID NO:4) 
(HisTag2-TNF.alpha.) is expressed in E. coli as described in example 2 and 
purified by metal chelate chromatography on an Ni.sup.2+ -NTA column: 
______________________________________ 
Matrix Ni.sup.2+ -NTA Agarose (Qiagen) 
Column 2 cm.sup.2 .times. 5 cm 
Buffer A 50 mM Tris-Cl, 300 mM NaCl, pH 8.0 
Buffer B 25 mM Bis-Tris-Cl, 300 mM NaCl, 10% glycerol, pH 
6.0 
Buffer C 25 mM Bis-Tris-Cl, 300 mM NaCl, 10% glycerol, 
1.0 M imidazole, pH 6.0 
Sample 10 ml E. coli extract in Buffer A 
Flow rate 60 ml per hour 
Fractions 2 ml (2 minutes) 
Elution 1. Sample (10 ml) 
2. 12 ml Buffer A 
3. 12 ml Buffer B 
4. 75 ml gradient from Buffer B to Buffer C 
5. 25 ml Buffer C 
______________________________________ 
To remove the purification tag comprising 11 amino acids, 
HisTag2-TNF.alpha. (0.5 mg/ml; pH6.0) is first treated with DAP I (50 
mU/mg) and GCT (1500 mU/mg) at 37.degree. C. for 30 minutes. The reaction 
is followed by SDS-PAGE (data not shown). It is seen that a small amount 
(about 15%) of unconverted HisTag2-TNF.alpha. is still present after 30 
minutes, and, prolonged treatment with DAP I and GCT does not change that. 
The reason for this could be either the presence of N-terminal terminal 
formyl methionine in a percentage of the protein or due to the known 
oligomeric structure of TNF.alpha.. After removal of the unconverted 
HisTag2-TNF.alpha. on a Ni-NTA column, the resultant product is subjected 
to N-terminal determination. No sequence is detected indicating the 
presence of pyroglutamine residue at the N-terminal of TNF.alpha.. To 
avoid uncontrolled further cleavage into TNF.alpha., DAP I is inactivated 
before treatment with PGAP. This is done by treatment at pH 10.5 for 10 
min, following by adjusting pH to 8.0. The presumed PyroGln-TNF.alpha. (at 
0.5 mg/ml and pH 8.0) is then treated for 60 min at 37.degree. C. with 
PGAP (500 mU/mg). PGAP is removed from the reaction mixture by IE-HPLC 
fractionation on MonoQ at pH 7.0, and the purified product is subjected to 
N-terminal sequence determination. The sequence is 
Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp SEQ ID NO: 9 in agreement with the 
N-terminal sequence of TNF.alpha.. 
EXAMPLE 4 
Generation of authentic TNF.alpha. from Met-Arg-6xHis- 
Ara-Gln-TNF.alpha.(HisTag3-TNF.alpha. SEQ ID NO: 10) 
(HisTag3-TNF.alpha.) is expressed in E. coli as described in example 2 for 
HisTag2-TNF.alpha. and purified by metal chelate chromatography on an 
Ni.sup.2+ -NTA column as described in example 3 for HisTag2-TNF.alpha.. To 
simplify the method and secure optimal removal of enzymes, a procedure is 
developed, which uses biotinylated DAP I and PGAP. These enzymes can be 
removed on streptavidin or Avidin matrices. Furthermore, GCT have affinity 
for Ni-NTA-Agarose and can therefore be removed simultaneously with 
unconverted HisTag protein. Alternatively, GCT can easily be removed on a 
cation exchanger at pH 7.5 because GCT has an isoelectric point around 
9.0. 
To remove the purification tag comprising 9 amino acids, HisTag3-TNF.alpha. 
(2.2 mg at 0.5 mg/ml; 4.5 ml) is treated with Biotin-DAP I (25 mu/mg) and 
GCT (750 mu/mg) for 30 min (Sample A). To remove Biotin-DAP I, sample A is 
passed through a 0.75 ml Streptavidin column (Sample B), and unconverted 
HisTag3-TNF.alpha. and GCT are removed on a Ni-NTA-Agarose column. (Sample 
C, PyroGln-TNF.alpha.). After removal of the unconverted 
HisTag3-TNF.alpha., the resultant product is subjected to N-terminal 
determination. No sequence is detected indicating the presence of 
pyroglutamine residue at the N-terminal of TNF.alpha.. 
To remove pyroGln 1.4 mg (6 ml) PyroGln-TNF.alpha. is treated with 
Biotin-PGAP (400 mu/mg for 18 h at 7.degree. C.; Sample D). To remove 
Biotin-PGAP, sample D is passed through a 0.75 ml Streptavidin column 
(Sample E). The purified product is subjected to N-terminal sequence 
determination. The sequence is Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp SEQ 
ID NO: 9 in agreement with the N-terminal sequence of TNF.alpha.. 
EXAMPLE 5 
Generation of authentic TNF.alpha. from Met-Lys-His-Leu-Ser-Glu- 
Ile-Phe-Glu-Thr-Met-Lys-Val-Glu-Leu-Ara-Gln-TNF.alpha.(BiotinTa91 
-TNF.alpha. SEQ ID NO: 11) 
BiotinTagl-TNF.alpha. is expressed in E. coli essentially as described in 
example 2 for HisTag2-TNF.alpha. and purified by affinity chromatography 
on an Immobilized Monomeric Avidin Agarose gel: 
______________________________________ 
Matrix Immobilized Monomeric Avidin Agarose (Pierce) 
Column 0.8 cm.sup.2 .times. 6 cm 
Buffer A 
100 mM sodium phosphate Na.sub.3 PO.sub.4, 150 mM NaCl, pH 
7.5 
Buffer B 
100 mM sodium phosphate Na.sub.3 PO.sub.4, 150 mM NaCl, 2 mM 
D-Biotin, pH 7.5 
Sample 10 ml E. coli extract in Buffer A 
Flow rate 
15 ml per hour 
Fractions 
1 ml (4 minutes) 
Elution 1. Sample (10 ml) 
2. 20 ml Buffer A 
3. 40 ml Buffer B 
______________________________________ 
To remove the purification tag comprising 17 amino acids, 
BiotinTagl-TNF.alpha. dialyzed against 20 mM sodium phosphate Na.sub.3 
PO.sub.4, 50 mM NaCl, pH 6.2 (2 mg at 0.5 mg/ml; 2 ml) is treated with 
Biotin-DAP I (100 mU/mg) and GCT (3000 mU/mg) for 60 min (Sample A). To 
remove Biotin-DAP I, unconverted BiotinTagl-TNF.alpha. and GCT, sample A 
is adjusted to pH 7.5 and passed through a 1.5 ml Avidin/0.5 
mlCM-Sepharose column equilibrated with 20 mM sodium phosphate Na.sub.3 
PO.sub.4, 50 mM NaCl, pH 7.5 (Sample B, PyroGln-TNF.alpha.). The resultant 
product is subjected to N-terminal determination. No sequence is detected 
indicating the presence of pyroglutamine residue at the N-terminal of 
TNF.alpha.. 
To remove pyroGln, 1.1 mg (4 ml) PyroGln-TNF.alpha. is treated with 
Biotin-PGAP (500 mU/mg for 18 h at 7.degree. C.; Sample C). To remove 
Biotin-PGAP, sample C is passed through a 0.75 ml Avidin column (Sample 
D). The purified product is subjected to N-terminal sequence 
determination. The sequence is Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp SEQ 
ID NO: 9 in agreement with the N-terminal sequence of TNF.alpha.. 
EXAMPLE 6 
Generation of authentic EGF from Met-Arg-6xHisGln-EGF SEQ ID NO: 12 
(HisTag3-EGF) 
(HisTag3-EGF) is expressed in E. coli essentially as described in example 2 
for HisTag2-TNF and purified by metal chelate chromatography on an Ni 
.sup.2+ -NTA column as described in example 3 for HisTag2-TNF.alpha.. 
To remove the purification tag comprising 9 amino acids, HisTag3-EGF (1.3 
mg at 0.5 mg/ml) is treated with Biotin-DAP I (50 mU/mg) and GCT (1500 
mU/mg) for 30 min (Sample A). To remove Biotin-DAP I, sample A is passed 
through a 0.75 ml Avidin column (Sample B,) and unconverted HisTag3-EGF 
and GCT is removed on a Ni-NTA-Agarose column. (Sample C, PyroGln-EGF). 
After removal of the unconverted HisTag2-EGF, the resultant product is 
subjected to N-terminal determination. No sequence is detected indicating 
the presence of pyroglutamine residue at the N-terminal of EGF. 
To remove pyroGln, 1.0 mg (6 ml) PyroGln-EGF is treated with Biotin-PGAP 
(1000 mU/mg for 18 h at 7.degree. C.; Sample D). To remove Biotin-PGAP, 
sample D is passed through a 0.75 ml Avidin column (Sample E). The 
purified product is subjected to N-terminal sequence determination. The 
sequence is Asn-Ser-Asp-Ser-Glu SEQ ID NO: 13 in agreement with the 
N-terminal sequence of EGF. 
EXAMPLE 7 
Generation of authentic EGF from 
Met-Lys-HisLeu-Ser-Glu-Ile-Phe-Glu-Thr-Met-Lys-Val-Glu-Leu-Ara-Gln-EGF 
(BiotinTagl-EGF SEQ ID NO: 14) 
BiotinTagl-EGF is expressed in E. coli essentially as described in example 
2 for HisTag2-TNF.alpha. and purified by affinity chromatography on an 
Immobilized Monomeric Avidin Agarose gel (Pierce) essentially as described 
for BiotinTagl-TNF.alpha. in example 5. 
To remove the purification tag comprising 17 amino acids, BiotinTagl-EGF 
dialyzed against 20 mM Na.sub.3 PO.sub.4, 50 mM NaCl, pH 6.2 (2.6 mg at 
0.5 mg/ml; 2 ml) is treated with Biotin-DAP I (250 mU/mg) and GCT (7500 
mU/mg) for 60 min (Sample A). To remove Biotin-DAP I, unconverted 
BiotinTagl-EGF and GCT, sample A is adjusted to pH 7.5 and passed through 
a 1.5 ml Avidin/0.5 mlCM-Sepharose column equilibrated with 20 mM sodium 
phosphate Na.sub.3 PO.sub.4, 50 mM NaCl, pH 7.5 (Sample B,-PyroGln-EGF). 
The resultant product is subjected to N-terminal determination. No 
sequence is detected indicating the presence of pyroglutamine residue at 
the N-terminal of EGF. 
To remove pyroGln, 2.1 mg (4 ml) PyroGln-EGF is treated with Biotin-PGAP 
(1000 mU/mg for 18 h at 7.degree. C.; Sample C). To remove Biotin-PGAP, 
sample C is passed through a 0.75 ml Avidin column (Sample D). The 
purified product is subjected to N-terminal sequence determination. The 
sequence is Asn-Ser-Asp-Ser-Glu SEQ ID NO: 13 in agreement with the 
N-terminal sequence of EGF. 
EXAMPLE 8 
Generation of authentic TNF.alpha. from 
Met-Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-His-Gln-His-Met-Asn-Ser-Ser-Arg-Gl 
n-TNF.alpha.SEQ ID NO: 15(S-Tagl-TNF.alpha.) 
(S-Tagl-TNF.alpha.) is expressed in E. coli essentially as described in 
example 2 for HisTag2-TNF and purified by affinity chromatography on 
immobilized S-protein (S-Protein Agarose Novagen) at 4.degree. C.: 10 ml 
E. coli extract containing S-Tagl-TNF.alpha. in 25 mM sodium phosphate 
Na.sub.3 PO.sub.4, pH 7.5, is added to 1.65 ml S-Protein Agarose 
equilibrated with 25 mM sodium phosphate Na.sub.3 PO.sub.4, pH 7.5, and 
the mixture is incubated for one hour at 4.degree. C., with mixing each 10 
min. The mixture is then packed in a small open plastic column (at 4 
.degree. C.) and after the gel has settled, it is washed with 5 ml 25 mM 
sodium phosphate Na.sub.3 PO.sub.4, pH 7.5. The column is closed at the 
bottom and heated to 37.degree. C. in a water bath, whereafter the 
S-Tagl-TNF.alpha. is eluted with 2.5 ml 25 mM sodium phosphate Na.sub.3 
PO.sub.4, 500 mM NaCl, pH 7.5, preheated to 37.degree. C. 
To remove the purification tag comprising 19 amino acids, S-Tagl-TNF.alpha. 
in 20 mM sodium phosphate Na.sub.3 /PO.sub.4, 50 mM NaCl, pH 6.2, (0.5 mg 
in 2.2ml) is treated with Biotin-DAP I (125 mU/mg) and GCT (2500 mU/mg) 
for 60 min (Sample A). To remove Biotin-DAP I and GCT, the sample is 
passed through a mixed 1.5 ml Avidin Agarose/0.5 ml CM-Sepharose column 
equilibrated with 20 mM sodium phosphate Na.sub.3 PO.sub.4, 50 mM NaCl, pH 
7.5, (Sample B), and unconverted S-Tagl-TNF.alpha. is removed on a 0.5 ml 
S-Protein Agarose column equilibrated with 20 mM sodium phosphate Na.sub.3 
PO.sub.4, 50 mM NaCl, pH 7.5, (4.degree. C.). (Sample C, 
PyroGln-TNF.alpha.). The resultant product is subjected to N-terminal 
determination. No sequence is detected indicating the presence of 
pyroglutamine residue at the N-terminal of TNF.alpha.. 
To remove pyroGln, 0.4 mg (7.5 ml) PyroGln-TNF.alpha. is treated with 
Biotin-PGAP (800 mU/mg for 16 h at 7.degree. C.; Sample D). To remove 
Biotin-PGAP, sample D is passed through a 0.75 ml Avidin column 
equilibrated with 20 mM sodium phosphate Na.sub.3 PO4, 50 mM NaCl, pH 7.5, 
(Sample E). The purified product is subjected to N-terminal sequence 
determination. The sequence is Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro SEQ ID NO: 
16 in agreement with the N-terminal sequence of TNF.alpha.. 
Example 9 
Generation of authentic TNF.alpha. from 
Met-Ara-Ser-Ala-Trp-Arq-His-Pro-Gln-Phe-Gly-Gln-TNF.alpha. 
(StrepTag1-TNF.alpha. SEQ ID NO: 17) 
(StrepTagl-TNF.alpha.) is expressed in E. Coli as described in example 2 
for HisTag2-TNF.alpha. and purified by affinity chromatography on 
Streptavidin Agarose: 
______________________________________ 
Matrix Streptavidin Agarose (Sigma) 
Column 0.8 cm.sup.2 .times. 6 cm 
Buffer A 
100 mM Na.sub.3 PO.sub.4, 150 mM NaCl, pH 7.5 
Buffer B 
100 mM Na.sub.3 PO.sub.4, 150 mM NaCl, 2 mM Imino-Biotin, pH 
7.5 
Sample 10 ml E. Coli extract in Buffer A 
Flow rate 
15 ml per hour 
Fractions 
1 ml (4 minutes) 
Elution 1. Sample (10 ml) 
2. 20 ml Buffer A 
3. 40 ml Buffer B 
______________________________________ 
To remove the purification tag comprising 12 amino acids, 
StrepTagl-TNF.alpha. in 100 mM Na.sub.3 PO.sub.4, 150 mM NaCl, pH 6.2, 
(2.8 mg in 2.8 ml) is treated with Biotin-DAP I (200 mU/mg), Biotin-APP 
(Biotinylated AAP; 75 mU/mg) and GCT (2500 mU/mg) for 90 min at 37.degree. 
C. (Sample A). To remove Biotin-DAP I, Biotin-APP, unconverted 
StrepTagl-TNF.alpha. and GCT, the sample is adjusted to pH 7.5 passed 
through a mixed 1.5 ml Streptavidin Agarose/0.5 ml CM-Sepharose column 
equilibrated with 20 mM Na.sub.3 PO.sub.4, 50 mM NaCl, pH 7.5, (Sample B, 
PyroGln-TNF.alpha.). The resultant product is subjected to N-terminal 
determination. No sequence is detected indicating the presence of 
pyroglutamine residue at the N-terminal of TNF.alpha.. 
To remove pyroGln, 0.4 mg (7.5 ml) PyroGln-TNF.alpha. is treated with 
Biotin-PGAP (600 mU/mg for 16 h at 7.degree. C.; Sample C). To remove 
Biotin-PGAP, sample D is passed through a 0.75 ml Avidin column 
equilibrated with 20 mM Na.sub.3 PO.sub.4, 50 mM NaCl, pH 7.5 (Sample D) 
The purified product is subjected to N-terminal sequence determination. 
The sequence is Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro SEQ ID NO: 16 in agreement 
with the N-terminal sequence of TNF.alpha.. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 17 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
HisSerGlnGlyThrPheThrSer 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Other 
(D) OTHER INFORMATION: Peptide attached to N terminus of 
glucagon. 
(A) NAME/KEY: Modified Base 
(D) OTHER INFORMATION: Xaa at position 1 represents 
des-His; Xaa at position 4 represents pGln 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
XaaSerGlnXaa 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GlyThrPheThrSer 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Other 
(D) OTHER INFORMATION: Peptide attached to N terminus of 
hTNFalpha. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetArgHisHisHisHisHisHisGlyArgGln 
1510 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CTGCAGCTAGCCAGGTCAGATCATCTTCCC30 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GGTGAATTCGGATCCTTA18 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CATGCGTCATCATCATCATCATCATGGGCG30 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Other 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CTAGCGCCCATGATGATGATGATGATGACG30 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ValArgSerSerSerArgThrProSerAsp 
1510 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
MetArgHisHisHisHisHisHisArgGln 
1510 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetLysHisLeuSerGluIlePheGluThrMetLysValGluLeuArg 
151015 
Gln 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Other 
(D) OTHER INFORMATION: Peptide attached to N terminus of 
EGF. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
MetArgHisHisHisHisHisHisGln 
15 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AsnSerAspSerGlu 
15 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
MetLysHisLeuSerGluIlePheGluThrMetLysValGluLeuArg 
151015 
Gln 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Other 
(D) OTHER INFORMATION: Peptide attached to N terminus of 
TNFalpha. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
MetLysGluThrAlaAlaAlaLysPheGluHisGlnHisMetAsnSer 
151015 
SerArgGln 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ValArgSerSerSerArgThrPro 
15 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Other 
(D) OTHER INFORMATION: Peptide attached to N terminus of 
TNFalpha. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
MetArgSerAlaTrpArgHisProGlnPheGlyGln 
1510 
__________________________________________________________________________