Separation of recombinant human interleukin-2 and methionyl interleukin-2 produced in Escherichiacoli

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SEPARATION OF RECOMBINANT HUMAN INTERLEUKIN-2 METHIONYL INTERLEUKIN-2 PRODUCED IN ESCHERICHIA

837-843

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Takao Yamada, Koichi Kato, Kenji Kawahara and Osamu Nishimura Biotechnology Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka 532, Japan Received

February

10,

1986

SUMMARY Escherichia &l harboring the gene coding for human interleukin-2 (IL-2) produced methionyl IL-2 (Met-IL-a) having an additional methionine residue at the amino terminus as well as IL-2 starting with the amino terminal alanine. IL-2 and Met-IL-2 were copurified from a cell-free extract. It was difficult to separate these two molecular speciesfrom each other becauseof the similarities of their physico-chemical characteristics. We found that the isoelectric points of IL-2 and Met-IL-2 were slightly but significantly different and succeeded in separating these two molecular species by utilizing the difference of their isoelectric points. The isoelectric points of IL-2 and Met-IL-2 thus obtained were determined to be 7.7 and 7.5, respectively. The in vitro specific activities of these two species were the same and similar to that of natural human IL-2 derived from peripheral blood lymphocytes. o 1986 Academic press, lnc. Interleukin-2 (IL-2) is a lymphokine produced by T-lymphocytes when they are stimulated by lectins or alloantigens (l-3).

It plays important roles in the

proliferation and differentiation of T-lymphocytes, as well as in the regulation of the immune systems. We succeededin cloning the human IL-2 gene derived from the activated peripheral blood lymphocytes and obtaining expression of the cloned gene in Escherichia @i (4). In the preceding paper (4), we described the purification and characterization of recombinant human IL-2 produced in g. &i

cells harboring the human

IL-2 gene. The amino acid composition, amino terminal amino acid sequenceand carboxyl terminal amino acid of the purified preparation were consistent with those deduced from the cDNA sequence. However, there existed a heterogeneity with’regard to the amino terminus. Besidesa molecular species with the amino terminal alanine (IL-2), the purified preparation contained another molecular

Met-IL-2, methionyl interleukin-2; Abbreviations used: IL-2, interleukin-2; SDS, sodium dodecyl sulfate; PTH, phenylthiohydantoin; HPLC, high performance liquid chromatography. 0006-291X/86

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species (Met-IL-2) of IL-2.

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having an additional methionine residue at the amino terminus

The additional

methionine

methionine codon designed to

be

residue

was derived

placed before the first

from

the initiator

alanine codon of the

mature natural IL-2 molecule (4). Recombinant additional initiator

proteins

methionine

residue

or separating

a protein

protein

methionylated

protein.

clinical

substantially

To our knowledge

activity,

stability

derivative.

free from its methionylated

protein derivative

in vivo and antigenicity from

of a

those of a nonfrom biological,

to obtain a recombinant

protein

derivative.

We report here that IL-2 and Met-IL-2

have different

that they can be separated by utilizing the difference

MATERIALS

to the

The physiological

it seems to be important

points of view

an

there have been no reports

could be different

Therefore,

often possess

corresponding

the amount of a methionylated

derivative

and pharmaceutical

system

amino termini

and its methionylated

such as biological

methionylated

by the & &i

at their

methionine codon (4-10).

dealing with either controlling

features

synthesized

isoelectric

points, and

of their isoelectric

points.

AND METHODS

Purification of recombinant human IL-2: Recombinant human IL-2 produced in Escherichia coli N4830/pTB285 cells harboring the human IL-2 gene was purified from a cell-free extract by cation exchange chromatography on CM-Toyopearl 650M (Toyo Soda Manufacturing Co., Ltd.) and reverse phase HPLC equipped with a TMS-250 column (Toyo Soda Manufacturing Co., Ltd.), as described previously (4). The purified preparation thus obtained was judged to be homogeneous on SDS-polyacrylamide gel electrophoresis, HPLC with two different systems and analytical ultracentrifugation (4). Assav of IL-2: IL-2 activity was determined by the ability to maintain an IL-2dependent murine cell line, NKC3, as reported previously (11, 12). Assav of protein: Protein was determined by the method of Lowry et al. (13) with bovine serum albumin as a standard. Mono P column, Pharmalyte (pH 8-10.5) and Polybuffer Chemicals and others: 96 were purchased from Pharmacia Fine Chemicals. Ultrapore RPSC column was from Altex. MicroPak SP C18-3 column was obtained from Varian Associates, Inc. Ampholine polyacrylamide gel plate was from LKB-Produkter.

RESULTS We have already shown that the purified preparation

of recombinant

human

IL-2 derived from E. coli was composed of two molecular species, IL-2 and MetIL-2 (4). The ratio of IL-2 : Met-IL-2

varied from lo:90 to 50:50 depending on the 838

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3

t-1

PH -9.0 -8.0

Figure 1. Gel isoelectric focusing of purified recombinant IL-Z, Pl and. P2. Isoelectric focusing was performed on an Ampholine polyacrylamide gel plate (pH 3.5-9.5) using a flat bed apparatus PBE 3000 (Pharmacia Fine Chemicals). The amounts of purified recombinant IL-2 (lane l), Pl (lane 2) and P2 (lane 3) used were 5 pg each. Electrophoresis was carried out at 4“C for 1.5 hr at a constant power of 30 W. The isoelectric point was determined by measuring pH of the solution extracted from the gel strips.

expression level and the culture conditions (data not shown). The chromatographic behaviors on gel filtration,

ion exchange chromatography, affinity

chromatography with anti-IL-P rabbit IgG and reverse phase HPLC for the two molecular species were indistinguishable and these attempts to separate them were unsuccessful. We were aware that the recombinant IL-2 preparation gave two clear bands on gel isoelectric focusing (Fig. 1, lane I). This result led us to consider that IL-2 and Met-IL-2 might possessdifferent isoelectric points. In order to clarify this point, a purified IL-2 preparation (composedof 25% IL-2 and 75% Met-IL-2, as estimated by the amino terminal amino acid analysis) was subjected to Mono Pfast protein liquid chromatography. The preparation gave two peaks, Pl and P2 (Fig. 2). The amino terminal the automated

Edman

amino acid sequencesof Pl and P2 as determined by

degradation

method 839

were

Ala-Pro-Thr-Ser-

and Met-Ala-

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----___ --._

E 1.0

-

-.._ .I.__

-

--__

--__

0

-----____

----___

9.0

-8.5

-7 r

- 8.0

; CL

z z 9 0.5

COMMUNICATIONS

-

: f! 51 90

0

5

10

Elution

15

20

volume

25

(ml)

Figure 2. Elution pattern of purified recombinant IL-2 on Mono P-fast protein liquid chromatography. A preparation containing purified recombinant IL-2 (5.9 mg) was applied to a Mono P column (0.5 x 20 cm) equilibrated with 25 mM diethanolamine-HCl (pH 9.4). Elution was performed with 1% (v/v) Pharmalyte (pH 8-10.5)- 5.2% (v/v) Polybuffer 96HCI (pH 8.0). Flow rate was 30 ml/hr. -----, pH. Fractions, Pl and P2, denoted by the bars were pooled A280; andabplied to reverse phase HPLC equipped with an Ultrapore RPSC column (4) in order to remove Pharmalyte and Polybuffer. Pl (1.1 mg) and P2 (2.9 mg) thus obtained were subjected to the protein-chemical analyses.

Pro-Thr-,

respectively

(TABLE

The carboxyl

I).

terminal

amino

acid of both

preparations as determined by the hydrazinolysis method (14) was threonine. The amino acid analysis showedthat Pl and P2 had the same amino acid composition except

that the number

of methionine

residues

per molecule

for Pl was 4.1 and

that for P2 5.1 (TABLE II). These results clearly indicate that Pl is IL-2 and P2 IL-2 and Met-IL-2 preparations obtained here were judged to be

is Met-IL-2.

substantially free from each other based on the amino terminal amino acid

TABLE

1. Amino terminal amino acid sequences of Pl and P2 PTH-amino

Cycle

acid detected (pmol) P2

Pl 1

Ala (2330)

2

3

4

Met (

Pro

21)

Ala (

Ala

(1670)

(

Thr ( 819)

(

Ser ( 222)

(

77)

Pro (

Pro 51)

33)

Thr (

Thr 40)

56)

45)

Ser (

16)

Met (2bOO)

Ala (2430)

Pro (1770)

Thr ( 789)

The sequence analysis was performed by the automated.Edman degradation method using a gas-phase protein sequencer model 470A (Applied Biosystems, Inc.). PTH derivatives of amino acids were determined by HPLC equipped with a MicroPak SP C18-3 column. The amounts of Pl and P2 used were 45 pg (3000 pmol) each.

840

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TABLE 11. Amino acid compositions of Pl and P2 Number of residues per molecule Amino acid Asp & Asn Thr Ser Glu & Gln Pro GUY Ala H-Cys Val Met He Leu Tyr Phe LYS His Ax Trp

Pla)

Pzd

11.8 n.6b) 7.5b) 18.6 5.3 2.2 4.9 2.W) 4.1 4.1 8.6 21.8 3.1 6.0 11.9 3.0 4.2 1.1

11.8 12.6b) 7.&l 18.7 5.3 2.2

Values predicted from cDNA sequence 12 13 8 18 5 2 5 3 4 4 9 22 3 8 11 3 4 1

EC) 4.1 5.1 8.6 21.9 3.2 6.1 11.9 3.0 4.2 1.1

The amino acid composition was determined on 24, 48 and 72 hr hydrolysates with 6 N HCl at llO°C in the presence of 4% thioglycolic acid. Amino acid analysis using ninhydrin was performed on a Hitachi amino acid analyzer model 835. a) Average of values on 24, 48 and 12 hr hydrolysates. b) Obtained by extrapolating to zero time of hydrolysis. c) Determined as cysteic acid on a 24 hr hydrolysate after performic acid oxidation.

sequence

It was also confirmed by rechromatography of IL-2 and Met-

analysis.

IL-2 on Mono P-fast protein liquid chromatography (data not shown). On gel isoelectric

focusing,

with

isoelectric

yi&

biological

Pl (IL-2)

points

and P2 (Met-IL-a)

of 7.7 and 7.5, respectively

activity

of IL-2

and Met-IL-Z,

gave a single

band of protein

(Fig. 1, lanes 2 and 3). The h as measured

by the ability

to

maintain NKC3 cells, was the same (38000f2000 U/mg of protein), which was in good accordance with that of natural human IL-2 purified from peripheral blood lymphocytes (11, 15).

DISCUSSION The presence

of methionine

at the amino terminus of recombinant human

IL-2 produced in E. coli is consistent with the evidence that formylmethionine initiates

protein

synthesis

in E. coli and that the formyl

group

is enzymatically

cleaved from the newly synthesized polypeptide chain (16-18). It is also known 841

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that such recombinant

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BIOPHYSICAL

proteins as human interferon-o

(7) and human growth hormone (8) synthesized an additional initiator

RESEARCH

methionine

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(5, 6), human interferon-y

by the E. coli system often possess

residue at their amino termini

corresponding

to the

methionine codon. Liang et al. (9) described that their recombinant

preparation plasmid

synthesized

in an E. coli

possessed

an additional

that

& &I

estimated

their

strain (C600) harboring’s

methionine system

pBR322-derived

at the amino terminus.

failed

to cleave

IL-2

They

the amino terminal

methionine becauie of the presence of a proline residue at position 2 of the IL-2 molecule. which

Contrary

to their observation,

may be responsible

from nascent proteins

However, lowered

(18), seemed to have worked

methionine

in our system

to give a

with alanine.

is a neutral

amino

acid

with

moder,ate

the addition of methionine at the amino terminus its isoelectric

aminopeptidase,

for the cleavage of the amino terminal

mature IL-2 molecule starting Methionine

methionine-specific

hydrophobicity.

of the IL-2 molecule

point by 0.2. The addition of an extra methionine

could

result in exposure of a hidden acidic amino acid residue or internalization

of a

basic amino acid residue near the amino terminal Such a conformational

change affecting

region of the IL-2 molecule.

the surface net charge of IL-2 might lead

to the unexpected acidic change of the isoelectric on the tertiary

structures

of IL-2 and Met-IL-2

on the change of the isoelectric We have described recombinant presented

study

Crystallographic

study

will provide a clear understanding

point.

here the first

protein and its methionylated in this

point.

might

example derivative.

be applicable

recombinant proteins and their methionylated

of mutually

separating

The technical

to the separation

a

approach of other

derivatives.

ACKNOWLEDGMENTS We thank Dr. Y. Sugino and Dr. A. Kakinuma of the Biotechnology Laboratories for their encouragement and discussion throughout this work. We are also grateful to Dr. 0. Shiho for the determination of lL-2 activity. Thanks are due to Mrs. S. Nakagawa for the amino acid analysis and Dr. J. R. Miller for reading the manuscript. 842

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