Chapter 11 The Interferons

433

Chapter 11 THE INTERFERONS SIDNEY PESTKA

DepaKhIent of Molecular Genetics and Microbiology. University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School Piscataway. New Jersey. U.S.A. 1 2

.

.

. 4.

INTRODUCTION

............................

............. . . .

PURIFICATION OF HUMAN LEUKOCYTE INTERFERON 2 . 1 Production. . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 High Performance Liquid Chromatography . . . . . . . . . . . . 2.3 Multiple Species of Leukocyte Interferon. . . . . . . . . . . 2.4 Amino Acid Sequences of Leukocyte Interferons . . . . . . . . 2.5 Carbohydrate Content . . . . . . . . . . . . . . . . . . . . . .

434 436 436 442 446 447

ISOLATION OF RECOMBINANTS .

447

8

.

..................... CONSTRUCTION OF EXPRESSION VECTORS . . . . . . . . . . . . . . . . . 4.1 Expression of HuIFN-u in E . coli . . . . . . . . . . . . . . . . 4.2 Expression of HuIFN-f3 in E . a. . . . . . . . . . . . . . . . 4.3 Other Expression Vectors.-. . . . . . . . . . . . . . . . . . PURIFICATION OF RECOMBINANT HUMAN LEUKOCYTE INTERFERON . . . . . . . LARGE SCALE IMMUNOAFFINITY CHROMATOGRAPHY. . . . . . . . . . . . . . 6 . 1 General Principles. . . . . . . . . . . . . . . . . . . . . . . 6.2 Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Aspects of Innnunoaffinity Chromatography . . . . . . . . . . . . PURIFICATION OF HUMAN FIBROBLAST INTERFERON. . . . . . . . . . . . . 7.1 Purification of Recombinant IFN-6 . . . . . . . . . . . . . . . PURIFICATION OF IMMUNE INTERFERON, IFN-Y . . . . . . . . . . . . . .

9

.

PURIFICATION OF ANIMAL INTERFERONS

3

5. 6

.

7.

434

8.1

....... .................

Immunoaffinity Chromatography of Recombinant IFN-y

........................ 11 . APPENDIX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 . CONCLUDING COMMJ3lTS

452 452 454 454 455 459 459 461 463 466 468 468 469 469 470 471 472

434

1.1-m

m y procedures have been described for the partial purification of human and

Although partial purification of the interferons as bands

animal

on SDS-polyacrylamide gels was reported by a number of groups, it

was

not until

1978 and thereafter that any interferon had been purified to homogeneity in solution in sufficient amounts for its chemical and physical characteri~ation.~-~ The introduction of reverse phase and normal phase high performance liquid chromatography to the purification of proteins""

lead to the first successful

purification of these proteins so that sufficient amounts were available in solution without detergent for their chemical, biological and innnunological studies. Various affinity purification techniques particularly antibody affinity chromatography were interferons.'

' lo-'

utilized

to

purify

human

leukocyte and

fibroblast

This review will concentrate on the purification of the

human leukocyte and fibroblast interferons from natural sources as well as the leukocyte interferon produced by recombinant DNA technology.

A

short review of

the purification of human ilmnune interferon is also included. 2.

RlRIFICATION OF HRWN -0CY'I'E

WERFEKN

Since their discovery, many attempts were made to purify the interferons with little success until recent years.

In fact, interferon used in experiments as

well as in initial human clinical trials was essentially a crude protein.fraction less than one percent of which by weight consisted of interferon. Because of the use of such crude interferon-containing material, it was not clear what activities of these preparations were indeed due inherently to the interferon present and what activities were due to the numerous other contaminating proteins.

By definition, the anti-viral activity was due to the interferon.

However, these crude preparations exhibited anti-protozoal and anti-bacterial

435

LEUKOCYTES

VIRUS (NDV)

INTERFERON

Fig 2-1 Production of interferon with Newcastle disease virus and leukocytes. Leukocytes, the white blood cells of peripheral blood, are obtained from normal donors or patients with chronic myelogenous leukemia. The cells are washed, placed in culture medium with Newcastle disease virus (or with Sendai virus), and incubated overnight at 37'C. Interferon is found in the culture medium after incubation. The cells and virus can be removed by centrifugation, leaving the culture medium containing interferon.

activities, inhibited cellular growth (anti-proliferative activity), blocked antibody synthesis, and were ascribed to have an enormous number of other activities. However, without essentially pure interferon, it was not possible to ,

demonstrate definitively whether or not a particular activity was due to the interferon protein molecule itself. Accordingly, it was essential to obtain pure interferon to determine what activities were an inherent part of the interferon molecule.

Since very little was known about the size and structure of the

interferons, the isolation of pure interferons would establish their chemical composition and structure as well as their biological activities. Some of the activities ascribed to interferon turned out to be exhibited by the pure forms, but many other properties were not demonstrable with pure preparations and were, therefore, due in whole or in part to one of the contaminants in the crude preparations. To obtain purified interferons, many groups began work on their purification shortly after their discovery.

436

2.1.

Production We began purification of interferon from human leukocytes in 1977.

This

interferon was produced by incubating human white blood cells with Newcastle disease virus or Sendai virus for 6-24 hr.20-23 The procedure was a combination of techniques that were previously reported.”’

25

The anti-viral activity was

found in the cell culture medium after overnight incubation of the leukocytes as illustrated in Fig. 2-1. We substituted milk casein for human or bovine serum in the culture medium as had been described.24 The use of casein, a single protein, instead of serum which contains many different and uncharacterized proteins, simplified the initial concentration and purification steps. We used leukocytes from normal donors as well as from patients with chronic myelagenous leukemia. These leukemic cells made large amounts of human leukocyte interferon when induced with Newcastle disease virus or Sendai virus.2” 2 6 - 2 8 The cytopathic effect inhibition assay for interferon as originally described took three days. Other assays for interferon were even longer. It was necessary

to develop a more rapid assay so that we could evaluate whether a purification step was useful.

A

cytopathic effect inhibition assay that could be done in

12-16 hr was developed2 ’

and accelerated the purification immensely.

2.2 High Performance Liquid Chrrnnatography For Protein Purification Because we knew that classical techniques for protein purification, as applied by others, were not remarkably successful in purification of the human interferons, we applied high-performance liquid chromatography (HPLC) to the purification of proteins.

Udenfriend, Stein and c~workers~’-~~ had developed

sensitive fluorescent techniques for detection of amino acids and peptides and had achieved the separation of peptides by reverse-phase HPLC. However, separation of proteins had not yet been accomplished. In the early experiments, there

437

was uniform failure to achieve any purification of proteins with reverse-phase HPLC because the interferon activity was continually lost.

At the time,

increasing ethanol concentration was used to elute proteins and this was tried for interferon and other proteins without success.

It was necessary to use a

less polar solvent to elute interferon. Although there was initial hesitancy to use I-propanol above 20% (v/v) and other organic solvents because of the limited solubility of proteins in such solvents, it was found that 9-propanol gradients effectively eluted interferon and other proteins without noticeable precipitation of the proteins at the concentrations

Furthermore, by changing

'

the pH of the elution buffer, a completely different separation could be achieved during elution of the same reverse-phase column with I-propanol. As subsequently demonstrated with fibroblast interfer~n,~ a large number

of different columns

and solvent systems could be used to effect resolution of proteins, By applying normal-phase chromatography with a diol silica column between the two reversephase columns, it was possible to use just three sequential HPLC steps to purify human leukocyte interferon to homogeneity. Sufficient amounts were purified in high yield for initial chemical characterization of the protein and for determination of amino acid composition. leukocyte interferon species

y2

The amino acid composition of the human was the first reported for any purified

interferon.' The initial steps included selective precipitations and gel filtration (Fig. 2-2)

followed by HPLC.

2-3) at pH 7.5

Did, Fig. 2-4),

The HPLC steps were reverse-phase chromatography (Fig.

on LiChrosorb RP-8, normal partition chromatography on LiCHrosorb and reverse-phase chromatography at pH 4.0 on LiChrosorb Re-8.

Gradients of q-propanol were used for elution of interferon from these columns (Figs. 2-3,

2-4,

2-5).

The overall purification was about 80,000-fold and the

specific activity of pure interferon was 2-4

x

10'

units/mg.'

Interferon

prepared by this procedure yielded a single band of M 17,500 on polyacrylamide gel electrophoresis. The anti-viral activity was associated with the single

438

LEUKOCYTES, NDV MEDIUM, CASEIN

r-

CELLS

I

PPT

I

12;;s

I

IDH4sup

1

11.5%TCASUP 1

I-s'4 uP% TCA

I

I

SUP

PPT--l GEL FILTRATION

I

HPLC 2-2 Flaw chart of initial steps in purification of leukocyte interferon. Cells and debris were removed by low speed centrifugation from the medium containing interferon. Casein was used as a serum substitute. By acidification of the medium to pH 4 with hydrochloric acid, the bulk of the casein, which precipitated, was removed from the interferon, which remained in solution. The interferon was concentrated by two steps, involving precipitation with trichloroacetic acid. The concentrated solution containing relatively crude interferon was separated into components of different sizes by gel filtration on Sephadex G l O O ia fhz3pf~senceof 4 M urea. Details of these procedures have been described. ' ' ' FIG.

protein band.5

The specific activity of this peak was 4 x lo8 units/mg and that

of some other leukocyte interferons are shown in Table 2-1. Several reports had previously described high-performance liquid chromatography of proteins, mainly on ion exchange and size exclusion

column^.^^'^^

However, those systems were either not colnmercially available or had a low capacity. With proper choice of eluent and pore size, octyl and octadecyl silica could be used for high resolution reverse-phase HPLC of both peptides and

439 REVERSE PHASE CHROMATOGRAPHY

I

I

HYORDPHOBIC POCKET OF PROTEIN BINDS TO OCTYL GROUPS

3. REMOVE PROTEINS ey ADDITION OF n-PROPANOL

High-performance liquid chromatography of leukocyte interferon on reverse-phase columns. Columns of porous silica to which octyl groups were bound were used to separate leukocyte interferons from other proteins and to resolve the individual leukocyte interferons. The proteins enter the interstices within the silica particles where the hydrophobic areas of the protein bind tightly to the octyl groups. The non-polar portion of n-propanol can interact with interferon, releasing interferon and other proEeins from the column. By eluting the proteins with an increasing gradient of n-propanol, the proteins are released in order of their increasing hydrophobicity. Those proteins binding most tightly are released only after high concentrations of 2-propanol are used. FIG. 2-3.

proteins.

Accordingly, with 2-propanol as eluent, the use of LiChrosorb RP-8

(octyl silica) columns for protein fractionation was a major factor in the success of the purification Fig. 2-5 A, C and D).

In addition, LiQlrosorb Diol,

which is chemically similiar to glycophase resins that have been used for exclusion chromatography of proteins, was introduced as a support for normal partition chromatography of proteins (Fig. 2-5

9).

High recoveries of interferon

440

PROTEIN BINDS TO DlOL GROUPS (PAIR OF OH GROUPS ON GLYCEROL BONDEO SILICA PARTICLES) IN HIGH n-PROPANOL CONCENTRATON.

RAOlON NUMBI

AT LOW n-PROPANM CONCENTRATION HYDROGEN BONDING TO WATER OCCURS. RELEASING PROTEIN FROM TUE COLUMN.

FIG. 2-4. Hydrogen-bonding high-performance liquid chromatography on diol-silica. Hydrogen bonding of proteins to columns containing glycerol covalently attached to silica were used to separate interferon species. Since hydrogen bonds &re relatively weak bonds and are readily made to the water in which the protein is dissolved, in order to force proteins to bind to the diol-silica, it was necessary to use a very high concentration of -n-propanol. Upon reduction of the n-propanol concentration, proteins are released from the c o l m in order -of their increasing ability to form H-bonds with the diol groups of the column. Those proteins released first form the weakest €I-bonds with the column. Multiple molecular species of human leukocyte interferon (a, 6, and y ) are shown in the figure. These designations should not be confused with the subsequent nomenclature of IE'N-a, IFN-6, and IFN-y assigned to leukocyte, fibroblast, and inunune interferons, respectively.

activity were obtained in each chromatographic step, a requirement when small amounts of initial starting Fterial are present.

Although the initial

experiments were performed with leukocytes from normal that leukocytes from patients with chronic myelogenous leukemia

it was found

(a), who were

441

Z

0

I U M IY -

z

ml

L

I

FRACTION NUMBER

2-5. High-performance liquid chromatography of interferon. (A) Chromatography on LiChrosorb RP-8 at pH 1.5. (B) Chromatography on LiChrosorb diol at pH 7.5. (C) Chromatography on LiChrosorb RP-8 at pH 4.0 of the y peak of part B. ( D ) Rechromatography on LiChrosorb RP-8 of the major activity peak of part C. The conditions were similar to those of step C. Several preparations carried through step C were pooled ( 1 3 X lo6 units) and applied to the last column. The gradations on the abscissa correspond to the end of the fractions. The solid61ines on the graph represent protein as measured by the fluorescarnine method. FIG.

442

undergoing leukapheresis to laver their peripheral white blood cell counts, were a rich source of interferon that appeared to be essentially identical to the human leukocyte interferon purified from leukocytes from normal donors.2'

As

with HPLC of interferon from normal leukocytes on the Diol column (Fig. 2-5 B), three major peaks of activity, labeled a, 8, Y*, were observed with interferon prepared from

CML

cells.

Although the protein profiles were almost identical,

the activity profiles showed that the amount of activity under peak in preparations from leukemic cells compared to normal leukocytes.' even from normal leukocytes, the ratio of peaks a, 8, and preparation to another.

y

12'

y

was 1-r

However ,

varied from one

Human lymphoblastoid interferon produced by suspension

cultures of Namalva cells was purified by a combination of immunoaffinity chromatography and other methods by Zoon g

$.13

The amino acid composition of

human leukocyte interferon purified by HPLC as described above6 shows similarity with human lymphoblastoid inte~feron'~ and one of the

types of mouse

interferon."

2.3.

Hultiple Species Of Leukocyte Interferon During the purification of leukocyte interferon, it became evident that

multiple species existed (Figs 2-4

and 2-5).

Human leukocyte interferon is

heterogeneous and several bands containing anti-viral activity ranging in t@~ from 15,000 to 21,000 are observed on SDS-polyacrylamide gel electroph~resis.~

*The natural interferons which were isolated from the mixture present in leukocyte interferon by high performance liquid chromatography5-' were then designated g l 5 , P1

8,, B,,

Y,,

y1

yJI

Y,,

Y,,

and 6.

Unfortunately, the

same terminology was later applied to designate leukocyte, fibroblast, and immune interferons, respectively as a, 8,

y.

TABLE 2-1

Specific Activities of Representative Purified Human Interferons

Relative titers Specific Activity (units/mg)

Source

IFN-a (Y, )

Leukocytes

4.0 X l o 8

MDBK

5-7

IFTS-u ( 5 )

Leukocytes

4.0 X 10'

MDBK

7

IFN-a (b3)

K G 1 cells

4.0 X l o 8

MDBK

37

IFN-a (Ly)

Namalwa cells

2.5

Human fibroblasts

13

IFN-aA

E. -coli -

3.0 X l o 8

MBDK

38,39

IFN-f3

Fibroblasts

GM-258

10,11

2-10

x lo8

x lo8

Assay cells

MDBK-1732

Interferon

RDBK/WISH

Reference

Fibroblasts

4.0 X 10'

-1732

9

IFN-f3

Fibroblasts

3.2 X 10'

A61732

8

IFN-f3

E. -coli -

9.4

E. -coli

1.2

1FN-v

Leukocytes

5.9

IFN-y

E. -coli -

1.0

IFN-8

[SEX'

,

] IFN-f3

x x

107

WISH

40

lo8

GEI-2504

41

x x

107

WISH

42,23

107

WISH

44 4

h W

444

Heterogeneity of human leukocyte interferon has also been observed by isoelectric

procedure^.^ ‘ 4 7 - 5 0

focusing46 and several types of chromatographic

As

noted

above, our initial work with high-performance liquid chromatography revealed three major groups of interferon species which were labeled a, f3, and

y

to their order of elution from a LiChrosorb Diol (polar-bonded phase)

according cO~UII~.~”

These groups were further resolved into several homgeneous components. Although others

had

reported

heterogeneity

in

crude

human

leukocyte

interferon

preparations ,4 7 - 5 0 this was not thought to be due to amino acid sequence heterogeneity.

In fact, it had been reported by a number of groups that leukocyte

interferon contained carbohydrate and that heterogeneity was due to differences in carbohydrate content of the protein.‘6’51-53

Thus, the well-established

heterogeneity of human leukocyte interferon was attributed to differences in the degree of glycosylation.

However, five purified species of leukocyte interferon

examined contained no detectable carbohydrate .7

We still have not examined all

the purified leukocyte interferon species isolated. content of each species analyzed

( a,

6, , 6, ,

f3,,

and

However, the amino sugar y, )

was determined to be

much less than one residue of either glucosamine or galactosamine per molecule of inte~feron.~ We therefore concluded that, contrary to general dogma, human leukocyte interferon is largely devoid of carbohydrate.

Nevertheless, we would

expect that some species exhibiting molecular weights significantly in excess of 19,000 would contain carbohydrate.

In general, these high molecular weight

species represent a small fraction of the natural human leukocyte interferons. Because

peptide

mapping

and

sequencing

revealed

significant

structural

differences among the species, we concluded that leukocyte interferon represents a family of homologous proteins. By analogous procedures, additional leukocyte interferon species were isolated from cultured myelobla~ts.~ ”5 4 Since our initial purification, other reportsl4 , 1 6 1 7 5 5 have also described multiple species of leukocyte interferon. I

I

Rllen and Fantes14 also found no carbohydrate on the species of leukocyte interferon they purified.

The dogma that interferons are glycoproteins has been

445 so

universal that leukocyte interferons are still called glycoproteins despite

the data to the contrary.

However, at least one minor species of leukocyte

interferon appears to be glycosylated although we are only now beginning to determine its carbohydrate content (see below).

HUMAN LEUKOCYTE INTERFERON QI, 1 10 20 CYS ASP LEU PRO GLN THR H I S SER LEU GLY SER ARC ARG THR LEU MET LEU LEU ALA GLN

Jr 21 30 40 MET ARG LYS I L E SER LEU PHE SER CYS LEU LYS ASF ARG H I S ASP PHE GLY PHE PRO GLN

-0

41 50 60 GLU GLU PHE GLY lrSN GLN PHE GLN LYS ALA GLU THR I L E PRO VAL LEU H I S GLU MET I C E

1

61 70 80 GLN GLN I L E PHE ASN LEU PHE SER TH? LYS ASP SER SER ALA ALA TRP ASP GLU THR LEU

81 90 100 LEU ASP LYS PHE TYR THR GLU LEU T Y R GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL I L E 101 110 120 GLN GLY VAL GLY VAL THR GLU THR PRO LEU MET LYS GLU ASP SER I L E LEU ALA VAL ARG

>

-

1

121 130 140 LYS TYR PHE GLN ARG I L E THR LEU TYR LEU LYS GLU LYS LYS TYR SER PRO CYS ALA TRP

0 150 155 141 GLU VAL VAL ARG ALA GLU I L E MET ARG SER PHE SER LEU SER THR

m--

Sequence of human leukocyte interferon g and B,. The bars under the sequence represent the tryptic peptides isolated and sequenced. Solid bars represent the sequence determined by Edman degradation of the peptides. Unfilled bars represent sequences that were consistent with the composition of the peptides and/or sequence assignments derived from the DNA of the correspon$&l recombinant. Details of these experiments have been The molecular weight of the protein as shown is 18,056. reported. ' FIG. 2-6.

446

2.4.

Amino Acid Sequences of Leukocyte Interferons Since only relatively small amounts of each species were isolated in these

early experiments, it was difficult to obtain information about their amino acid sequence.

The first determinations of the amino acid sequences of the

amino-terminal ends of human interferons were by Zoon interferon, and by Knight

g 9J.”

g 1.56 for a leukocyte

for human fibroblast interferon.

Several

reported the amino-terminal sequence of another human months later, Levy & 3.58 leukocyte interferon; and Stein

g

aJ.,’

Okamura

g

&.,I5

and Friesen g

the amino-terminal sequence of human fibroblast interferon.

&.,’

It was clear that

all the amino acid sequences obtained for human fibroblast interferon were identical and that we had all purified and sequenced the same protein.

It was

comforting to know that the new microsequencing procedures were dependable. However, it was striking that the amino-terminal sequence of the human leukocyte interferon species g , g and BlS0 was different from that of the leukocyte There were two differences in the sequence. 4..56

interferon reported by Zoon

Because the differences involved amino acids that would not be expected to cause errors, we felt that both sequences were correct and that the difference dramatically confirmed the fact that the leukocyte interferons consist of a family of closely related proteins. We subsequently isolated a human interferon DNA

the coding sequence of which was virtually identical to the

sequence of our pure proteins. Thereafter, Levy &

&.60

and Shively g

g.61 reported amino acid

of three species of human leukocyte interferon.

So

sequences

far, the sequences of two

leukocyte interferons were determined almost completely (Fig. 2-6). Additional sequences were reported by

Allen and Fantes14 reported the sequences of

tryptic fragments obtained from a mixture of several leukocyte interferon species.

All these sequences are sufficiently different to establish very

clearly the concept of a family of closely related proteins. above, 4

1

59

As described

the first clone of human leukocyte interferon isolated in our

laboratory was almost identical in sequence to human leukocyte interferons g and

447

S, species, which were however, ten amino acids shorter than expected from the

DNA sequence. It should be noted that during this time several groups12’1*‘62-65

reported the purification of mouse interferons, and some amino acid sequences were reported.66

2.5.

Carbohydrate Content The carbohydrate content of all the species of human leukocyte interferon

(IFN-a) which have been derived from patients with chronic myelogenous leukemia ((3%)and

purified to homogeneity has now been determined.67 Amino sugar content

was measured by high performance liquid chromatography and detection of acid hydrolysates of each sample. amounts of glucosamine.

fluorescamine

Two species showed significant.

Most of the purified species of leukocyte interferon

from a myeloblast cell line were also tested and two species were found to contain sugar residues.

These forms also differed from the

CML

interferons in

that they revealed the presence of greater amounts of galactosamine.

The

apparent lack of carbohydrate in some of the higher molecular weight species of interferon implicated factors other than glycosylation in the molecular weight differences. The results indicated that some species of IFN-a are glycosylated to various degrees.

3.

ISOLATICW OF RECCMBIN?WIS

Because recombinant DNA technology offered an opportunity to produce large amounts of HuIFNs economically, many scientific teams set out to clone them in bacteria.

Several groups achieved the isolation of recombinants for several

HuIE‘N-a species5

’

and for I F N - $ ~ * ~ ~ - ’achieving * their goals by somewhat

different but analogous approaches. The cloning and expression of HuIFN-a as an illustration of these procedures is described. Isolating HuIF” DNA sequences was a formidable task since it meant preparing DNA

recombinants from cellular mRNA that was present at a low level.

This task

had never been accomplished previously from a protein whose structure was

448

unknown.

In addition, in order to reconstruct DNA recombinants which would

express natural IFN, it is useful to know the partial amino acid sequence of the proteins, particularly at the M1,-

and COOH-terminal ends.

Without this

information synthesis of natural HuIFN in bacterial cells would not have been possible.

Thus, purification of the HuIFNs and determination of their

structure5,617.13,14

I

37.58.60,61

assisted us and others in these efforts.

To isolate recombinants containing the human DNA corresponding to IFN-a, we used a number of procedures. Firstly, it was necessary to isolate and measure the IFN mRNA.

This was accomplished several years earlier when IFN mRNA was

translated in cell-free extracts (Fig. 3-1)73r74and in frog o o ~ y t e s . ~ ~ -The ~’ next step was to prepare sufficient mRNA from cells synthesizing IFN, and this was accomplished with both fibroblasts and A

leukocyte^.'^'

library of complementary DNA (cDNA)was prepared from a template of part-

ially purified

isolated from human leukocytes synthesizing IFN (Fig. 3-2).

mRNA

The dC-tailed double-stranded(ds) DNA obtained was hybridized to dGtailed DNA from plasmid pBR322 which had been cleaved at the =I and

introduced into Escherichia

coli

by

restriction nuclease site

transformation.

About

14,000

tetracycline-resistant and ampicillin-sensitive transformants were obtained. This provided a large group of c m recombinants with DNA copies of all the mRNAs extracted from the leukocytes. The next and the hardest part of the procedures was to find in this vast library of recombinant plasmids those which contained

DNA encoding IFN.

If we had been able to begin with pure IFN ~RNA, this would

have been a simple task. However, we did not have pure IFN

mRNA.

We began with

a mixture of mfWA molecules and only relatively few were IFN-specific. we could show their presence by translating the activity (Fig. 3-1). procedure.

and assaying the products for IFN

In this situation, we had to devise an indirect two-stage

In the first stage, we screened all the bacterial colonies to find

those with cDNA made from the RNA of induced cells; among these there might have been some carrying IFN cDNA.

We, therefore, screened all the recombinants for

their ability to bind to mRNA from cells synthesizing IFN (induced cells), but

449

not to mRNA from uninduced cells (those not producing IFN) (Fig. 3 - 3 ) .

To do

this, individual transformant colonies were screened by colony hybridization for the presence of induced-specific sequences with 32~-labeledIFN mRNA induced cells) as probe.

( ~ R N Afrom

In the presence of excess mRNA from uninduced cells

(Fig. 3 - 3 ) , recombinants that were representative of mRNA sequences existing only in induced cells should be evident on hybridization.

This screening procedure

allowed us to discard about 90% of the colonies: since their plasmids carried no induced cDNA, these could not encode IF".'

'

In the second stage, we had to identify those recombinants containing the IFN DNA sequences among the remaining 10%.

To do this, we pooled the recombinant

plasmids in groups of ten and examined these for the presence of IFN-specific sequences by an assay which depends upon hybridization of IFN mRNA to plasmid DNA*59.82

Plasmid DNA from ten recombinants was isolated and covalently bound to

diazobenzyloxymethyl (DBM) paper (Fig. 3 - 4 ) .

The

mRNA

from induced cells was

hybridized to each filter. Unhybridized mRNA was removed by washing. After the

0

CELL-FREE INTERFERON SYNTHESIS

PHENOL EXTRACTION OLIGO(dT)-CELLULOSE

POLY rI POLY r C CYCLOHEXIM IDE

EX TRACT

FIG. 3-1. IFN synthesis in a cell-free system. Human fibroblasts were stimulated to produce IFN by treatment with the inducer p0lyI:polyC. The total RNA present was extracted 4 hr later from the cell with phenol, and the fraction enriched in mRNA was selected on an oligo(dT)-cellulose column. An S-30 supernatant translation fraction was prepared from mouse cells. This was able to translate the various mRNA species, including that for IFN-6, into the corresponding proteins. The IFN formed was detected in a standard antiviral assay. In this way, biologically active HuIF" was synthesized in the test tube for the first time. By injecting the mRNA into intact frog oocytes, the mRNA could be measured at, 1/100 to 1/1000 the levels that could be detected in the cell-free extracts.

450

0

12SmRNA

3

5-'--An

0 pBR322

@ mRNA cDNA

HYBRID

1 0 IM NaOH

@

+

cDNA

5'

Restriction Endonuclease

DNA Polymerase I (Klenow Fraament)

@

ds cDNA HAIRPIN

+

mmmmr;'

3

3'

I

@

Terrnirnl Deoxynucleotidyl Tronsferase. dGTP

k ~ , : b "Oeoxynucleotidyl "l Tronsferose. @

3'CCCCCC 5'mmmrmzCCCCC

3'

v 3' GGGGGG

5'

GGGGGG3'

@

cDNA Insert

FIGURE 3-2. Preparation of IFN DNA recombinants from mRNA. Step 1 - a human leukocyte suspension was induced to form IFN by stimulation with Sendai virus. An extract was made 6 hr later from which mRNA was prepared that was enriched in 12s mRNA by differential centrifugation. Step 2 - the mRNA fraction was used as a template with AMV reverse transcriptase in the presence of all four deoxynucleotide triphosphates and oligo(dT),,-,, as a primer to generate a DNA stand complementary to the mRNA (cDNA) forming mRNA:cDNA hybrids. Step 3 - treatment with 0.1 M NaOH digested away the RNA, leaving a cDNA fragment with a selfannealed 3' end. Step 4 - extension of the chain with DNA polymerase (Klenow fragment) produced as ds-cDNA containing a hairpin loop. Step 5 - treatment with nuclease S1 opened the hairpin loop. Step 6 - the ds-cDNA was sized on an 8% polyacrylamide gel followed by electroelution to give a fraction containing at Step 7 - treatment with terminal deoxynucleotidyl least 500 base pairs. transferase in the presence of dCTP added cytosine homopolymer tails at the 3' ends of each strand. Step 8 - the plasmid pBR322 was cleaved (linearized)at the PstI site with PstI restriction endonuclease. Step 9 - homopolymer tails of ngodeoxyguanyls were added to the 3' ends of the linearized plasmid DNA with terminal deoxynucleotidyl transferase in the presence of dGTP. Step 10 - the two fragments with complementary sticky ends were mixed in equal proportions and annealed to yield a recombinant plasmid. A larger number of similar plasmids not containing IF" genetic information were also formed during this procedure. This cDNA library contained copies of all the mRNA in these cells including IFNspecific mRNA.

451

specifically hybridized mRNA was eluted, both fractions were translated in Xenopus laevis oocytes. Once a positive group had been found (one in which the specifically hybridized mRNA yielded IFN after microinjection into frog oocytes), it was necessary to identify the specific clone or clones containing IF" cDNA. The ten individual colonies were grown, the plasmid

DNAS

were prepared, and each

individual DNA was examined by mRNA hybridization as above (Fig. 3 - 4 ) .

By these

procedures a recombinant, plasmid 104 (p104), containing most of the coding sequence for a HuIFN-a was identified.59 The

DNA

sequence was determined and

found to correspond to what was then known of the amino acid sequence of purified HuIFN-a

.

58'60

The cDNA insert in p104 contains the sequence corresponding to

more than 80% of the amino acids in IFN-a, but not for those at its aminoterminal end. It was, therefore, used as a probe for finding a full-length copy of the IFN cDNA sequence which could be used for expression of HuIFN-a in _E.

coli. In addition, p104

DNA

was used to isolate DNA sequences corresponding to

other IFN-u species directly from a human gene bank. Examination of the coding regions of the leukocyte IFN genes that have been isolated in our laboratory and others have shown that these correspond to a family of homologous proteins, the IF"-a

specie^^"^

which are closely related to

each other and yet each unique in amino acid sequence.

Thus, the previously

discovered heterogeneity in HuIFN-a was shown to be at least in part the result of distinct genes representing each expressed HuIFN-a sequence.

The cloned

HuIFN-UA, which was the first one we isolated, corresponds to the natural IFNs which we purified from the mixture present in IFN-a by HPLC and termed g and The g

Bl.

and 6, indicated here refer to two different leukocyte IFN fractions (see

footnote on page 000). By similar procedures to those described for p104, plOl was shown to contain the sequence for HuIFN-6. -6 were identified.

Thus the nucleotide sequences coding for HuIFN-a and

452

CLONE SCREEN1NG : HYBR IDIZ AT ION

x + y LABELED m R N * x~ EXCESS ~ ~ ~ UNLABELED ~ 1

mRNAIND*

,

I

6 C 0L0NY HY BRI DIZ AT ION

00000 0.000 00000 FIG. 3-3. Schematic outline of hybridization procedure. E. coli cells were transformed with the recombinant plasmids generated as describeGin Fig. 3-2. Colonies derived from Individual transforme3$ bacteria cells were transferred to filter paper, fixed and probed with a P-labeled mRNA preparation from cells producing IFN and, therefore, enriched in IFN mRNA ( x + y). The hybridization was performed in the presence of excess unlabeled mRNA from uninduced cells ( x ) . This procedure identified bacterial colonies containing DNA coding for the protein specifically formed in induced leukocytes, including IE". The induced-specific sequences (y) incJude IFN sequences as well as others that are induced concomitantly with IE".

4.

4.1.

C f 3 l S ~ IOF~EXPRESSION VEClORS

Expression of H u I W a in _E. i& AS

noted above, p104 contained most of the sequence for a HuIFN-o, but did

not contain the sequence coding for the amino terminus of the protein. Accordingly, p104 was used to screen additional cDNA recombinants, and a number of cDNA recombinants which hybridized to its unique IFN-coding sequence were identified.84 Most contained IF" DNA sequences of sufficient size to code for an entire IF"-a protein. Our first full-length recombinant isolated corresponded to IFN-aA,59' 8 4 and its entire E t I insert was sequenced.

Existing knowledge about IFN protein

453 CLONE SCREENING: BINDING ACTIVE mRNA

PLASMID DNA

BOUND mRNA

UNBOUND mRNA

OOCYTE ASSAY

FIG. 3-4. Schematic illustration of screening of recombinants with DBM paper. Plasmid DNA was isolated from a pool of colonies, e.g., ten colonies as shown in the figure, and bound covalently to DBM paper. To detect a recombinant containing an IFN sequence (shown as a zigzag in the illustration), mRNA prepared from cells synthesizing IF" was hybridized to the bound DNA. After hybridized mRNA had been removed by washing, the specifically hybridized mRNA was eluted and translated in Xeno us laevis one oocytes. By this procedure, those pools of clones in which*east recombinant contained an IF" sequence were identified. Thereafter, individual clones in each pool were separately screened by the same procedure. Unbound mRNA was microinjected into oocytes as well as a control to ascertain that the mRNA was not destroyed by the manipulations. sequences permitted us to determine the correct translational reading frame, and, hence, to predict the entire amino acid sequence for the IF" species encoded by this recombinant. The mature IF"-aA was expressed directly by reconstruction of the re~ombinant.'~ The leader sequence of the protein was removed and an

ATG

translation-initiation codon was placed immediately preceding the codon for the first amino acid of mature IFN-aA.

Next, a 300-base pair E o R I fragment of E.

coli DNA was constructed, containing the tryptophan (9) promoter-operator and the Irp leader ribosome-binding site, but stopping short of the needed to initiate translation of the leader peptide of the

ATG

sequence

9 regulatory

region. This DNA fragment was attached to the reconstructed HuIFN-aA in front of the ATG codon (Fig. 4-1).

Inserted into _E.

g, the

recombinant yielded high

levels of activity, about 2 X 10' units (about 1 mg) of IFN per liter of culture. The IFN protein produced in _E.

behaves similarly to IF" formed directly by

human leukocytes: it is stable to acid treatment, is neutralized by antiserum to

454

HuIFN-a, and binds to monoclonal antibodies specific for HuIFN-u.

It can be

purified to homogeneity with the use of monoclonal antibodies.38‘ ”

With

improvements in fermentation and in the bacterial strains, high levels of HuIFN-a (1 X

lo1’ units/l, 50

mg/l) can be obtained in large scale cultures.

Similar

high levels have been produced with other IFNs and other eukaryotic proteins.

4.2

Expression of HuIEN-B in _E. As

already mentioned,

identified.’ ‘

‘

a

coli bacterial

clone

containing

IFN-f3

DNA

was

In contrast to the multiple species of I F N - a , experiments have

indicated that there is only a single HuIFN-f3 gene which corresponds to this recombinant. To express mature HuIFN-f3 directly .!ni

G ,a

series of plasmids

which placed the synthesis of the 166-amino-acid polypeptide under

promoter

control (Fig. 4-1) were constructed. The IFN-8 produced in bacteria is similar to that formed by human fibroblasts by several criteria.

It contains the same

amino acid sequences, has the same relative antiviral activity on human and bovine cells, and is neutralized by rabbit antibodies to IF”+ antibodies to HuIFN-a.

but not by

However, IFN-f3 made in bacteria is not glycosylated,

‘lo whereas that made by human cells contains ~arbohydrate.~

4.3

Other Expression Vectors

Although _E.

coli

has been the predominant host used for the production of

recombinant proteins, other host-vector systems have been studied and may be used more in the future. vectors

.’

* O6

For example, the HuIFNs have been expressed in yeast

In addition, the regulatory regions of eukaryotic viruses have

been utilized to obtain expression of IFNs.’’-’*

Furthermore, by direct

transformation of cells with the HuIFN-8 gene together with the gene for dihydrofolate reductase resistance, the HuIFN-f3 gene has been amplified in Chinese hamster cells.93 With the use of such eukaryotic vectors, glycoslated HuIFN-f3 has been produced at high levels.

455

REGULATION OF INTERFERON EXPRESSION n + TRYPTOPHAN PROMOTER

P

a

0

INTERFERON CODING REGION

I

INTERFERON

FIG. 4-1. Regulation of IPN expression in E. coli. A 300-base pair fragment of E. coli DNA was prepared with the r e g t r i a n endonuclease EcoRI. This containedthe Q promoter-operator and ribosome-binding site.-The complete DNA sequence coding for IFN-UA was prepared, and an ATG codon (which is the signal for the initiation of translation of the mRNA into a protein) was placed immediately in front of the codon for the first amino acid of the mature protein. The EcoRI Q promoter-operator fragment was ligated distal to this ATG codon andinserted into plasmids. Bacteria transformed with a plasmid containing this control region and the IFN-coding sequence formed IFN Leukocyte IF"-& expression was thus in large amounts in the absence of 9. promoter-operator . 4 regulated by the 5.

HJRIFICATION OF I "

HUMAN LEuRo(NTE-1

Monoclonal antibiodies to the human leukocyte interferons were used to purify recombinant human leukocyte A interferon ( IFN-UA) produced in ba~teria.~ *' coli containing the human leukocyte interferon, IFN-A, were broken.

E. -

Unbroken

cells and cellular debris were removed by centrifugation. The IFN-UA and soluble bacterial proteins remained in the cell lysate. Nucleic acids

(DNA

and RNR) that

made the lysate viscous and thus difficult to handle easily were precipitated by combination with polymin P.

The soluble proteins remaining in the lysate can

be concentrated, or without concentration, passed directly through a column containing a monoclonal antibody to human leukocyte interferon (Fig. 5-1).

The

antibodies bound only the interferon; all the other components and proteins

456

PURIFICATION OF INTERFERON WITH MONOCLONAL ANTIBODIES

FIG. 5-1. Schematic illustration of purification of interferon with monoclonal antibodies. Monoclonal antibody is attached covalently to a solid support. For the puri3fai,cf$ion of IFN-UA, monoclonal antibody LI-8 was coupled to Affi-gel 10. The bacterial extract containing IFN-UA is passed through the monoclonal antibody column (1). IFN-uA, but not other proteins, bind to the column which is exhaustively washed to remove other contaminating proteins (2). Purified IFN-aA is then eluted by adjusting the elution buffer to pH 2.5 with acetic acid ( 3 ) . passed through the column. After washing the column, the IFN-UA bound to the column was removed by elution with an acidic soluti~n.~*"~A virtually pure interferon solution is eluted from the monoclonal antibody column (Figs. 5-2 and 5-3).

The column was then washed and neutralized so that it could be used

repeatedly. The interferon solution was concentrated by passage over a column of

carboxymethyl-cellulose.

'

The activity of the purified interferon made in

bacteria was very similar to that of the same human leukocyte interferon species synthesized by human cells. Since repeated use of the monoclonal antibody columns is possible, these affinity columns provide a convenient method for preparing homogeneous human leukocyte interferon from bacterial fermentations.

Interferon prepared by

modifications of these procedures is now being used in clinical trials in humans.

457

I

I

I

I

I

I

I

1

I

I

3.0-

;? X

.-Ei c 0

e 2.0 q .-u) c

-

a

2 W LL

!-

1.0

z

-

-

high salt

1

pH2.5

1

FRACTION NUMBER

Purification of interferon by monoclonal antibody inmunoadsorbent column chromatograph%. Experimental details are sumMrized in the text and given e ~ ~ e ~ f i e r e . ~ "The ~ column consisting of monoclonal antibody LI-8 attached to Affi-gel 10 was washed sequentially with several buffers. Fractions 127-130 containing interferon activity were eluted with 0.2 N acetic acid, 0.15 M NaCl, 0.1% Triton X-100, pH 2.5. Almost all of the protein appeared in the fl&&rough fraction of the column. Interferon was measured by radioinnnunoassay. FIG. 5-2

Several biological activities of this purified recombinant interferon have been determined.

The recombinant

IFN-aA

exhibits anti-viral activity and anti-

proliferative activity comparable to those of crude and purified natural leukocyte

interferon^.^

IFN-d

also stimulates natural killer cell activityg6

as do the natural species of human leukocyte interfer~n.~' With the eventual availability of large amounts of homogeneous IFN-uA, extensive clinical trials, biological studies, and determination of its structure are achievable.

458

Rapid advances in recombinant DNA technology have made it possible to produce virtually an unlimited supply.

As

noted by McGregor and Ramelfg8 worldwide

demand for interferon as a therapeutic agent will likely be measured in the kilogram range but not in tons as indicated for antibiotics.

5-3 Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified interferon. The gel was stained with 2.5% Coomassie Brilliant Blue. Approximately 20 ~g of interferon purified by monoclonal FIG.

antibody column LI-8 was subjected to electrophoresis. Stds, represents the standard molecular weight markers; E. coli, represents total proteins from Escherichia coli 530; LI-8, represent thefraction after passage through the monoclonal antibody column; and CM-52, represents the purified interferon after passage through the CM-52 column. Data from Staehelin e J $.38'39

459

Expression of interferon in bacterial cells and subsequent purification with the use of imnobilized monoclonal antibodies as shown above is essentially a one step purification scheme. When combined with high expression in bacteria, this procedure provides high recoveries and low cost of the interferon. scaled-up purification of recombinant leukocyte

A

Below a

interferon with immobilized

monoclonal antibodies is described. This purified material is being investigated in clinical studies for efficacy as an anti-viral and/or anti-tumor agent.99

6.1 General principles McGregor and Rmelg8, Tarnowskiloo and Dunnill'ol

have considered the

large-scale recovery and containment of recombinant proteins destined for clinical application.

In our goal to obtain a pure homogeneous product,

purification procedures were developed that would minimize steps and provide a high yield and purity.

In principle, immunoaffinity chromatography as described

above on the laboratory scale can be used.

Simply, the antibodies or antigens

are chemically coupled to an inert solid matrix and subsequently crude samples containing the antigen or antibody to be purified are passed over a column of the imnobilized protein.

Other components of the crude sample can be washed free

from the column and the absorbed antigens or antibodies subsequently recovered by elution with a suitable dissociating agent.

The greater the specificity of the

immobilized component (the monoclonal antibody), the greater the likelihood of developing a rapid and convenient purification. Eveleigh'O

*

has noted that the general acceptance o f inmunoaffinity chromato-

graphy has been rather slow and that the potential of the technique has yet to be fully realized.

Recently, however, imunoaffinity techniques have been gaining

460

popularity

.’

O ’-lo

zoon

&.13

utilized antibody affinity chromatography in

their multi-step procedure to purify human leukocyte interferon from Namalva lyrnphoblastoid cells.

Berg

isolated 13 species of leukocyte

@.lrl’

interferon in essentially one step by inununoaffinity chromatography with plyclonal antibodies to human leukocyte interferon. As

noted above, Staehelin et al.3ar’9,107p108 used hybridoma techniques for

production of monoclonal antibodies that were subsequently immobilized and used to purify IFN-aA

from bacterial cells.

They had identified 13 hybridomas

secreting monoclonal antibodies against human leukocyte interferon.

These

hybridomas were subsequently grown in mice and large amounts of monoclonal antibodies were recovered from acidic fluid. One of the hybridomas, designated LI-8,

was a particularly stable cell line and was used for routine production of

monoclonal antibodies. Subsequently, the monoclonal antibodies were purified and irmnobilized on activated cross-linked agarose, Affi-gel 10.’’ crude _E.

coli extracts containing IFN-UA

’

Relatively

were passed over a column of immobilized

LI-8 monoclonal antibody after which desorption with dilute acetic acid resulted

in a 1200-fold purification with at least 95% recovery. With the exception of a small amount of dimeric IFN-aA, the interferon appeared homogeneous when analyzed on denaturing gel electrophoresis. Establishing stable hybridomas that secrete monoclonal antibody can be a very tedious and expensive undertaking. established, monoclonal

antibodies

However, once hybridoma cell cultures are offer

certain

advantages

over

serum

antibodies. They are monospecific; they provide a homogeneous interaction with the antigen; they can be chosen for unique properties such as stability in certain reagents or for low affinity to make elution of the antigen more efficient; hybridomas can produce homogeneous antibodies reproducibly; and in general the yield of monoclonal antibodies is much greater than plyclonal

461

antibodies obtained from serum. Even with these important advantages, the use of monoclonal antibodies for the large-scale purification of proteins presents a serious supply problem. This problem was recognized by Eveleighio2 One solution is to automate the purification process.

Anderson

g

e.'Og designed

a

preparative inarmnoaffinity system which was programmed by mechanical switches. with the advent of commercial microprocessors, it is relatively convenient to automate immunoaffinity chromatography for the purification of IFN-aA with the use of electronic controls.

Basically, the procedure includes a column of

immobilized LI-8 monoclonal antibody interfaced with a time-based microprocessor controlling solution absorption, washing, and elution from the column.

Control

with a time-based microprocessor is economical and versatile.

6.2

-1es Specific procedures for large-scale fermentations, cell extraction, and

preparation of the inmunoabsorbent gel have been described by Tarnowski and Liptak'"

and Tarnowski

& .'I1

Briefly, bacteria were harvested during

logarithmic growth by centrifugation and the bacterial cell pellets were stored at -20°C until used.

All fermentations carrying the plasmid pIFN-UA and

procedures in the purification were performed in accordance with the Z H Guidelines for Recombinant DNA Research.

Frozen cell pellets were suspended in

lysis buffer and passed twice through a Manton-Gaulin homogenizer at 7000 p.s.i.

38,39

Polyethyleneimine

(Polymin P) at pH 7.9 was added to the crude

lysate to a final concentration of 0.35% (w/v) to facilitate nucleic acid and cell debris removal.

The lysate was then centrifuged and the supernatant sub-

sequently obtained was reduced to approximately one-half to one-forth volume by

462

ultrafiltration on a Millipore Pellicon unit equipped with 50 ft2 of PTGC membrane (10,000 m~co)''~

Alternatively, cells could be extracted directly with

2 M guanidine hydrochloride as described by Tarnowski & As

&.'"

noted above, Affi-gel 10 was utilized for the laboratory scale purificaHowever, insufficient availability of Affi-gel 10 for production

tion of IFN-UA.

scale applications prompted development of an alternative gel which afforded the same performance. Nishikawa and Bailon" of Sepharose

48

*

synthesized the aminopropyl derivative

and extended the ligand structure by succinylation of amino

groups to produce agarose bearing 5-acyloxysuccinimido groups (P. Bailon and A. €I. Nishikawa,

personal communication).

The coupling reaction was carried out

producing an antibody density of 11-13 mg of protein per m l of gel.'"

Although

the density of this gel was only about half that reported by Staehelin

a1.,38,39it did not affect the performance of the column. The binding capacity of interferon on this innnuno- adsorbent was approximately 1 mg of IFN-aA per ml of gel. The IFN-ah purification steps were essentially as described above,'*' however,

modifications

implemented.l 1

'

for

practical

considerations

in

scale-up

39

were

Ion exchange chromatography of the innnunoabsorbent-purified

IFN-UA was performed on carboxymethylcellulose as des~ribed.~~ '" This step was

essential for the removal of the non-ionic detergent Triton X-100. Table 6-1 summarizes the purification of IFN-aA after 15 cumulative elutions of the antibody c o l m .

At each cycle, sufficient concentrated E. @ extract

was loaded onto the column to achieve complete saturation with IFN-aA.

was 73% with a specific activity of 2 x 10'

Recovery

units/mg of protein. The IFN-CA was

purified approximately 160-fold and appeared to be homogeneous when analyzed by SDS-polyacrylamide gel electrophoresis as above. The difference in purification

463

level (157- vs 1200-fold) between those data'" et

g . 3 8 r 3 9

process.

and those reported by Staehelin

reflects improvements both in expression and in the fermentation Specific activity of the purified IFN-UA in comparison to other

leukocyte interferon species is given in Table 2-1.

6.3

Aspects of Immmoaffinity Chromatography Purification of human leukocyte interferon synthesized in bacteria was

accomplished by employing these inmobilized monoclonal antibodies.

Utilization

of this essentially, one-step purification procedure provided the first pure human interferon for clinical evaluation and on January 15, 1981, material produced at HoffmaM-La Roche Inc. was used in a patient for the first time. The development of technology from the laboratory level to a prototype operation for comrcial production was essential to provide sufficient material for continued large-scale clinical trials. Since sufficient antibody supplies were a limiting factor, it was imperative to develop a system whereby a column of immobilized monoclonal antibody could be used repeatedly and the longevity evaluated. After several hundred cycles, the capacity of the column for interferon was not diminished.

There were, however, detectable levels of mouse inununoglobulin in

each antibody column elution which was removed below detectable levels in the subsequent purification steps.1 1 1 , 1 1 3

Several thousand column cycles would be

required before a decrease in IFN-aA yield would be observed through loss of antibody. The use of dilute acetic acid as an eluting agent does not appear to be particularly denaturing to the imbilized antibody. Contamination with

-E. coli proteins does not appear to

be a serious problem.

Success with the

imoaffinity purification of IFN-uA has prompted the extension of similar purification procedures to other proteins and other affinity chromatography

464

Table 6-1. Sununary of Large Scale Purification of IFN-d Total Volume Step

Total

(liters) units

Polyethyleneimine

137

2.6 X 1OI2

16.5

1.9 X 10l2

Specific

protein activity Recovery Purification (g)

(unitshg)

(%)

1.4 X l o 6

100

1.0

2.2 X 10'

73

157

1863

factor

supernatant Antibody eluate

8.7

Adapted from Tarnowski and Liptakl"

procedures. Nevertheless, non-inarmnoaffinity procedures have also been successful.

The use of a series of chromatographic procedures without inmunoaffinity

chromatography to purify IFN-a2 produced in _E.

& was described by Thatcher and

Panayotatos.' Inarmnosorbents made by covalently coupling monoclonal antibodies to agarose gels have high specificities and therefore are powerful tools for the purification of proteins.

Specificity was demonstrated in the case of IE'N-d:

essentially crude extracts of _E.

coli are passed

over a column of inununoabsorbent

gel and IF"-d is purified in essentially one step.

Recovery yields are high

after this step, but the formation of IFN-d oligomers via intermolecular disulfide bonds' l 5 requires additional steps in the purification scheme to obtain purified monomers.'

l1

The lower specific activity of oligomers and the

possibility they might cause adverse reactions in human clinical trials make it desirable to obtain a monomer preparation.

465

The monomer preparation contains at least two molecular forms, slow-moving monomer (SMMM)and fast-mving monomer

(m). Ten monomer

forms are predicted if

all the possible combinations of disulfide bonds and free sulfhydryl forms are considered.

Radiolabeling the preparation with the alkylating agent N-ethyl

maleimide showed that The free -SH in

SMM

SMM

contained free sulfhydryl groups but

EM ' M

did not."'

potentially could give rise to oligorners on storage. It is

assumed that FMM contains two disulfide bonds.

Separation of these two species

is desirable to obtain a stable product form. Bodo and Fogy116 isolated at least seven monomer forms of IFN-dC(Arg)

( identical

to [Arg2

,

Arg3 ] IFN-A) by

affinity purification and reverse-phase chromatography. Subsequent characterization of these component monomers showed that disulfide bonds were partially and completely reduced, scrambled, and correctly formed between cysteine residues 1-98 and 29-138.

They concluded, as we have, that these various species may

arise from the reduced monomer. The formation and removal of IFN-uA oligomers adversely impacts on the overall recovery of the purification process. Since most oligomers are disulfide linked, and pH-dependent polymerizations can be controlled, conversion to fully active monomer has been demonstrated with reduction-oxidation systems."'

'

11'

Incorporation of a redox system into the purification scheme could make the process one of extraordinary yield. The high homology among the leukocyte interferon a subtypes engenders an epitope on many that is recognized by monoclonal antibody LI-8. other recombinant interferons, IFN-aA/D 1FN-d

(sII), IFN-d, IFN-aD,

Consequently, IFN-d,

IFN-61,

and IFN-d have been purified by procedures similar to the process

described he re. Despite initial skepticism, monoclonal antibody immunosorbent chromatography has proven to be a viable procedure for the purification of proteins for pharma-

466

ceutical applications. The ability to re-use the same column hundreds of times makes these columns practical because the quantity of monoclonal antibody required

is

reasonable

and

readily

obtainable

without

heroic

efforts.

Accordingly, the commercial manufacture of protein therapeutics by immobilized monoclonal antibodies for purification should be considered when planning the research and development of these pharmaceuticals.

7. RlRIFICATION OF

€lIPUW FI-WT

" 1

Several laboratories reported the purification and partial structural analysis of human fibroblast interferon with the use of SDS-polyacrylamide gel electrophoresis as the last step in purification."

'11

To obtain a product free

of salt and solvent, we developed a simple two-step purification of fibroblast interferon.'

The first step in the purification from the crude interferon-

containing medium involved Blue-Sepharose chromatography, a procedure described previously.49'5 7 on octyl silica.

The second step involved high performance liquid chromatography The amino acid composition and 19 residues of the amino-

terminal sequence of human fibroblast interferon were determined.' was identical to the first 13 residues reported by Knight

_ et _ al.15

and the first ten residues reported by Friesen 5

The sequence and Okamura

&.57

e.'

The Blue-Sepharose step (Fig. 7-1) provides a high purification factor, but results in a dilute solution of interferon in 50% ethylene glycol.

The final

product is then obtained in concentrated form free of ethylene glycol and buffer salts by HPLC (Fig. 7-2).

Since only volatile eluents are used, the interferon

may be recovered salt and solvent free simply by evaporation.

The specific

activity of the purified protein is 3 X 10' units/mg of protein (Table 2-1), similar to that of the purified human leukocyte interferon species. Representative specific activities of various purified natural and recombinant 1m-B

467 CHROMATOGRAPHY OFCRUDE INTERFERON ON BLUE SEPHAROSE CL-68

70

FRACTION

7-1. Blue-Sepharose chroaratography of crude human fibroblast interferon. Fractions 1-9 are eluted with 30% ethylene g)ycol, whereas the remaining fractions are eluted with 50% ethylene glycol. The volume per fraction is 25 ml.; 0, interferon; A, protein.

FIG.

TIME (minutes)

7-2 High-performance liquid chromatography of human fibroblast interferon. About 10' units of interferon were applied to an Rp-8 column. A step gradient of increasing p-propanol at pH 4 . 2 was pumped a t 22 ml/hr. Interferon was eluted in a broad peak toward the end of the 32% c-propanol step. A portion (3%) of the $elm effluent was monitored with fluorescamine for determination of protein. FIG.

468

It should be noted that the last step

preparations are summarized in Table 2-1.

and Berthold et al."

in the purification employed by Knight"

consisted of

SDS-polyacrylamide gel electrophoresis.

7.1

Purification of Recombinant 1-6

By analogous procedures, IFN-8 produced in _E.

coli has

been p~rified.~' _E.

coli cells were broken with a Manton-Gaulin homogenizer. Most of the IFN-f3 was associated with the sedimentable cell debris which was extracted with 7 M guanidine hydrochloride.

Blue-Sepharose chromatography was then employed to

purify IFN-8 as described above.

coli producing

Purification of [Ser1"1IFN-f3 from _E.

this analogue of IFN-8

was accomplished by sonication of cells followed by extraction of cellular debris with 2% SDS.41

The [Ser"']IFN-f3

was then purified by successive steps of

extraction with 2-butanol, acid precipitation, and gel filtration (Sephacryl S-200 and Sephadex 6 7 5 chromatography). Unlike human leukocyte interferon, which has been isolated as several different

species,6,7,14,16,1~~2~,3~,54

species has so far been isolated.

only a single fibroblast interferon

Human fibroblast and leukocyte interferons

appear to bind to the same cell receptor'"

and have similar activities.

Some

homology is evident from comparison of the DNA sequences coding for these interferons.

8.

PURIFICATION OF IMMUNE ,-I

IE'N--y

Natural IFW-y was purified by several groups with the use of similar procedures.42,43,118-125

The use of controlled-pore glass, concanavalin

A-

469

Sepharose, and gel filtration were generally used by most laboratories.

The

procedures vary from laboratory to laboratory as described in detail in the above reports.

Natural IFN-y is glycosylated.

'

'

Specific activity of the

'

purified IFN-y was about 6 x lo7 units/mg (Table 2-1). Although IFN-y appears as monomers of MW of 20,000 and 25,000 on SDSpolyacrylamide gel electrophoresis,' 2 o ' ' 2 2 ' '

the functional unit of

IF"-y

appears to have a target size equivalent to the tetramer.126

8.1

Itmunoaffinity Chromatography of Recombinant IFN-y Recombinant IFN-y produced in

5. & could be extracted from cells by

sonication or directly with 7 M guanidine hydrochloride.

This extract, after

absorption and elution from silica, was then chromatographed on a column containing a monoclonal antibody reacting with the carboxy-terminal end of IFN-Y.~'

The

specific activity of the purified product was 1

x lo7

of the purified natural protein (Table 2-1).

Similar procedures could be used

for natural IFN-y.

units/mg comparable to that

Although the monomer of IFN-y produced in

5. coli has a

MW

about 17,1100,~ * * I 2 7 the target size of the functional unit also appears to be a tetramer.' * 9.

PURIFICATICW OF A N I m

This review does not summarize the extensive work carried out on many animal interferons from manrmalian species as well as avian species. For the production and purification of these interferons, the reader should consult reviews noted above as well as specific articles describing these.1-3'"8'127-129

470

Although purification of the interferons to homogeneity remained elusive about two decades after their discovery, they are now available in pure form in many laboratories. _E.

e.For

The largest amounts available are the species produced in

purification, immunoaffinity chromatography has proven to be

generally useful both on a laboratory as well as a commercial scale. Nevertheless, techniques other than inmunoaffinity chromatography have proven to be successful as well.

The availability of these proteins for laboratory and

clinical studies has already catalyzed extensive new developments with these agents.

In the years ahead, it is likely our understanding and use of these

agents will be even more expansive and provide new insights into their actions.

-

Conceivably, we many begin to understand the physiological roles of the interferons as well.

I thank

Ms.

Dawn Foster for her assistance in composing the manuscript.

47 1

11. APPENDIX

1. LiChrosorb RP-8 (EM Laboratories, Elmsfor, NY) LiChrosorb Diol. (EM Laboratories, Elmsford, NY) 2.

Sephadex (Pharmacia Fine Chemicals, Piscataway, NJ)

3.

Polyacrylamide G e l (Numerous sources for acrylamide)

4.

Diazo benzyloxymethyl paper (see Ref. 59 and 82 for preparation)

5.

Polymin P (Polyethyleneimine) (Sigma Chemical Co., St. Louis, MO)

6.

Affi-gel (Bio Rad Laboratories, Rockville Center, NY)

7.

Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ)

8.

PTGC membrane (Millipore Corp., Bedford, MA)

9. Carboxymethyl cellulose (Whatman Inc., Clifton, NJ) 10. LI-8 Monoclonal Antibiody (See Ref. 38)

11. Blue Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) 12. Manton - Gaulin Homogenizer (Gaulin Corp., Everett, Mass)

13. Conconavalin - A Sepharose (Sigma Chemical Co., St. Louis, MO)

472

12.

REFWENCES

1. K. Berg, Acta Path. Microbiol. Scand., Section C, Suppl. 279, (1982) 1-136. 2. S. Pestka, Meth. Enzymol. 78, (1981a), 1-632. 3.

S. Pestka, Meth. Enzymol. 78, (1981a), 1-677.

4. S. Pestka, Arch. Biochem. Biophys. 221, (1983), 1-37. 5. M. Rubinstein, S. Rubinstein, P.C. Familletti, M.S. Gross, R.S. Miller, A.A. Waldman, and S. Pestka, Science 202, (1978), 1289-1290 6. M. Rubinstein, S. Rubinstein, P.C. Familletti, R.S. Miller, A.A. Waldman, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 76, (1979), 640-644.

7.

M. Rubinstein, W.P.

Levy, J.A. Moschera, C.-Y.

Lai, R.D.

Hershberg, R.T.

Bartlett, and S. Pestka, Archs Biochem. Biophys. 210, (1981), 307-318.

8.

S. Stein, C. KeMy, H.-J.

Friesen, J. Shively, U. Del Valle, and S. Pestka,

Proc. Natl. Acad. Sci U.S.A. 77, (1980), 5716-5719. 9. H.-J.

Friesen, S. Stein, M. Evinger, P.C.

Familletti, J. Moschera, J.

Meienhofer, J. Shively, and S. Pestka, Arch. Biochem. Biophys., 206, (1981), 432-450 10.

E. Knight, Jr., Proc. Natl. Acad. Sci. U.S.A., 73, (1976), 520-523.

11. W. Berthold, C. Tan, Y.H. Tan, J. Biol. Chem. 253, (1978), 5206-5212. 12. B. Cabrer, H. Taira, R.J. Broeze, T.D. Kempe, K. Williams, W.H. Slattery, W.H. Konigsberg, and P. Lengyel, J. Biol. Chem. 254, (1979), 3681-3684. 13. K.C. Zoon, M.E. Smith, P.J. Bridgen, D. Zur Nedden, and C.B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 76, (1979), 5601-5605. 14. G. Allen, and F.H. Fantes, Nature (London), 287, (1980), 408-411. 15. H. Okamura, W. Berthold, L. Hood, M. Hunakpiller, M. Inoue, H. SmithJohannsen, and Y.H. Tan, Biochemistry 19, (1981), 3831-3835. 16. K.C. Zoon, Meth. Enzymol. 78, (1981), 457-464. 17. K. Berg, and I. Heron, K d 78, (1981), 487-499.

473

18. Y. Kawade, J. F’ujisawa, S. Yonehara, Y. Iwakura, and Y. Yamamoto, K d 78, (1981), 522-535. 19. E. Knight, Jr. and D. Fahey, J. Interferon Res. 2, (1982), 421-429. 20. P.C. Familletti, and S. Pestka, Antimicrob. Ag. Chemother. 20, (1980), 1-4. 21. P.C. Familletti, R. McCandliss, and S. Pestka, K d 20, (1981), 5-9. 22. A.A. Waldman, R.S. Miller, P.C. Familletti, S. Rubinstein, and S. Pestka, Meth. Enzymol. 78, (1981), 39-44. 23. R.D. Hershberg, E.G. Gusciora, P.C. Familletti, S. Rubinstein, C.A. Rose, and S. Pestka, X d 78, (1981), 45-48. 24.

K. Cantell, and D.R. Tovell, Appl. Microbiol. 22, (1971), 625-628.

25. E . F . Wheelock, J. Bacteriol. 92, (1966), 1415-1421. 26. S.H.S. Lee, C.E. Van Rooyen, and R.L. Ozere, Cancer Res. 29, (1969), 645-652. 27. G.Y. Hadhazy, L. Gergely, F.E. Toth, and G.Y. Szegedi, Acta Microbiol. Acad. Sci. Hung. 14, (1967), 391-397. 28. M. Rubinstein, S. Rubinstein, P.C. Familletti, L.D. Brink, R.D. Hershberg, J. Gutterman, J. Hester, and S. Pestka,

In:

Peptides:

Structure and

Biological Function, pp. 99-103, Gross, E. and Meienhofer, J. (eds) Pierce Chemical Company, Rockford, Illinois. 29. P.C. Familletti, S. Rubinstein, and S. Pestka, Meth. Enzymol. 78,

1981),

387-394. 30. S. Rubinstein, P.C. Familletti, and S. Pestka, J. Virol. 37, (1981), 755-758. 31.

S. Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M.

Weigele, Science 178, (1972), 871-872. 32. S. Stein, P. Bohlen, J. Stone, W. Dairman, and S. Udenfriend, Archs Biochem. Biophys. 155, (1973), 203-212. 33. P. Bohlen, S. Stein, J. Stone, and S. Udenfriend, Analyt. Biochem. 67, (1975), 438-445.

474

34. M. Rubinstein, and S. Pestka, Meth. Enzymol. 78, (19811, 464-472. 35.

F.E. Regnier, and R. Noel, J. Chromatogr. Sci. 14, (1976), 316-320.

36.

S.-H. Chang, R. Noel, and F.E. Regnier, Analyt. Chem. 48, (19761, 1839-1845.

37. D.S. Hobbs, and S. Pestka, J. Biol. Chem. 257, (1982), 4071-4076. 38. T. Staehelin, D.S. Hobbs, H.-F. Kunq, and S. Pestka, J. Biol. Chem. 256, (19811, 9750-9754. 39. T. Staehelin, D . S .

Hobbs, H.-F.

Kung, and S. Pestka, in Methods in

Enzymology. Vol. 78, Interferons, Part A. S. Colowick, and S. Pestka, Eds., Academic Press, New York, 1981, 505-512. 40.

J.A. Moschera, D. Woehle, K.P. Tsai, C.-H. Chen, and S.J. Tarnowski, Meth. Enzym01.

119, (1986), 177-183.

41.

L.S. Lin, R. Yamamoto, and R.J. Drmond, K d , 119, (1986), 183-192.

42.

I.A. Braude, Prep. Biochem. 13, (1983), 177-190.

43.

I.A. Braude, Biochemistry 23, (1984), 5603-5609.

44. H.-F. Kung, Y.-C.E.

Pan, J. Moschera, K. Tsai, E. Bekesi, M. Chang, H.

Sugino, Meth. Enzymol. 119, (1986), 204-210. 45. W.E. Stewart, 11, Virology 61, (1974), 80-86. 46. W.E. Stewart, 11, L.S. Lin, M. Wiranowska-Stewart, and K. Cantell, Proc.

Natl. Acad. Sci. U.S.A. 74, (1977), 4200-4204. 47 *

E.T. Torma, and K. Paucker, J. Biol. Chem. 251, (1976), 4810-4816.

48.

J.K. Chen, W.J. Jankowski, J.A. O'Malley, E. Sulkowski, and W.A. Carter, J. Virol. 19, (1976), 425-434.

49. W.J.

Jankowski, W. Von Muenchhausen, E.

Sulkowski, and W.A.

Carter,

Biochemistry 15, (1976), 5182-5187. 50.

P.M. Grob, and K.C. Chadha, ibid 18, (1979), 5782-5786.

51.

S. Bose, D. Gurari-Rotman, U. Th. Ruegg, K. Corley, and C.B. Anfinsen, J.

Biol. Chem. 251, (1976), 1659-1662.

475

52.

S.

53.

P.J. Bridgen, C.B. Anfinsen, L. Corley, S. Bose, K.C. Zaon, U. Ih. Ruegg, and

Bose, and J. Hickman, K d 252, (1977), 8336-8337.

C.E. Buckler, K d 252, (1977), 6585-6587. 54.

D.S. Hobbs, J. Moschera, W.P. Levy, and S. Pestka, Meth. Enzymol. 78, (1981), 472-481.

55. K.C. Zoon, In:

The Biology of the Interferon System, pp. 41-55, De Maeyer,

E. Galasso, G. and Schellekens, H. (eds) Elsevierflorth Holland, Amsterdam. 56.

K.C. Zoon, M.E. Smith, P.J. Bridgen, C.B. Anfinsen, M.W. Hunkapiller, and L.E. Hood, Science 207, (1980), 527-528.

57. E. Knight, Jr., M.W. Hunkapiller, B.D. Korant, R.W.F. Hardy, and L.E. Hood,

Science 207, ( 1980), 525-526. 58. W.P. Levy, J. Shively, M. Rubinstein, U. Del Valle, and S. Pestka, PKOC.

Natl. Acad. Sci. U.S.A. 77, (1980), 5102-5104. 59.

S. Maeda, R. McCandliss, M. GKOSS, A. Sloma, P.C. Familletti, J.M. TabOK,

M. Evinger, W.P. Levy, and

S.

Pestka, Proc. Natl. &ad.

Sci. U.S.A. 77,

(1980), 7010-7013; ibid 78, (19811, 4648. ~

60. W.P. Levy, M. Rubinstein, J. Shively, U. Del Valle, C.-Y. Lai, J. Moschera,

L. Brink, L. Gerber, S. Stein, and S. Pestka, K d 78, (198l), 6186-6190. 61. J.E. Shively, U. D e l Valle, R. Blacher, D. Hawke, W.P. Levy, M. Rubinstein, S.

Stein, W.C. Mffiregor, J. Tarnowski, R. Barlett, D. Lee, and

S.

Pestka,

Anal. Biochem., 126, (1982), 318-326. 62. Y.

Iwakura, S. Yonehaca, and Y. Kawade, J. Biol. Chem. 253, (1978),

5074-5079. 63.

M. Kawakita, B. Cabrer, H. Taira, M. Rebello, E. Slattery, H. Weideli, and P. Lengyel, J. Biol. Chem. 253, (1978), 598-602.

64. J. De Maeyer-Guignard, M.G. Tovey, I. Gresser, and E. De Maeyer, Nature,

London, 27, (19781, 622-625.

476

65. J. De Maeyer-Guignard, Meth. Enzymol. 78, (19811, 513-522. 66. H. Taira, R.J.

Broeze, B.M.

Jayaram, P. Lengyel, M.W.

Hunkapiller, and

L.E. Hood, Science 207, (1980), 528-530. 67. J.E. Labdon, K.D. Gibson, S. Sun, and S. Pestka, Arch Biochem. Biophys. 232, (19841, 422-426.

68. S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. Ecscdi, W. Boll, K. Cantell, and C. Weissmann, Nature (London), 284, (1980), 316-320. 69. T. Taniguchi, S. Ohno, Y. Fujii-Kuriyama, and M. Muranwtsu, Gene, 10,(1980), 11-15. 70.

R. Derynck, J. Content, E. De Clercq, G. Volckaert, J. Tavernier, R. Devos, and W. Fiers, Nature (London), 285, (19801, 542-547.

71. M. Houghton, A.G. Steward, S.M. Doel, J.S. Emtage, M.A.W.

Eaton, J.S. Smith,

T.P. Patel, H.M. Lewis, A.G. Porter, J.R. Birch, T. Cartwright, and N.H. Carey, Nucleic Acids Res., 8, (1980) 1913-1931. 72.

D.V. Goeddel, H.M. Sheppard, E. Yelverton, D. Leung, R. Crea, A. Sloma, and S. Pestka, Nucleic Acids Res., 8, (1980) 4057-4074.

73.

S. Pestka, J. MCIMeS, E.A. Havell, and J. Vilcek, Proc. Natl. Acad. Sci.

U.S.A.,

72, (19751, 3898-3901.

74. M.N. Thang, D.C. Thang, E. De Maeyer, and L. Montagnier, Proc. Natl. Acad. Sci. U.S.A.,

72, (19751, 3975-3977.

75. F.H. Reynolds, Jr., E. Premkumar, and P.M. Pitha, Proc. Natl. Acad. Sci. U.S.A.,

72, (19751, 4881-4885.

76. R.L. Cavalieri, E.A. Havell, J. Vilcek, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A.,

74, (19771, 3287-3291.

77. R.L. Cavalieri, E.A. Havell, J. Vilcek, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A., 78.

74, (1977), 4415-4419.

R.L. Cavalieri, and S. Pestka, Tex. Rep. Biol. Med., 35, (1977), 117-123.

417

79. R. McCandliss, A. Sloma, and S. Pestka, in Methods in Enzymology, Vol. 79, Interferons, Part B, Pestka, 80.

S.,

Ed., Academic Press, New York, 1981, 51-59.

P.C. Familletti, R. McCandliss, and S . Pestka, Antimicrob. Agents Chemother., 20, (1981), 5-9.

81.

S.

Maeda, M. Gross, and

Interferons, Part

9,

Pestka, in Methods in Enzymology. Vol. 79,

S.

Pestka,

S.,

Ed., Academic Press, New York, 1981,

613-618. 82. R. McCandliss, A. Sloma, and

S.

Interferons, Part B, Pestka,

Pestka, in Methods in Enzymology, Vol. 79, S.,

Ed., Academic Press, New York, 1981,

618-622. 83. S. Pestka, Sci. A m . , 249(2), 1983, 36-43.

84. D.V. Goeddel, E. Yelverton, A. Ullrich, H.L. Heyneker, G. Miozzari, W. Holmes, P.H. Seeburg, T. Dull, L. May, N. Stebbing, R. R.

McCandliss, A. Sloma, J.M. Tabor, M. Gross, P.C.

Crea, S. Maeda,

Familletti, and S.

Pestka, Nature (London), 287, (1980), 411-416. 85.

R.A.

Hitzeman, D.W. Leung, L.J. Perry, W.J. Kohr, H.L. Levine, and D.V.

Goeddel, Science, 219, (1983), 620-625. 86. M.D. Schaber, T.M. DeChiara, and R.A. Kramer, in Methods in Enzymology, VOl . 119, Interferons, Part C, Pestka,

S.,

Ed., Academic Press, New York,

986,

416-423.

87. K. Z i M , P. Mellon, M. Ptashne, and T. Maniatis, Proc. Natl. Acad. Sci. U.S.A., 79, (1982), 4897-4901. 88.

D. Canaani, and P. Berg, Proc. Natl. Acad.

Sci. U.S.A.,

79, (1982),

5166-5170. 89. R. Devos, H. Cheroutre, Y. Taya, W. Degrave, H. Van Heuverswyn, and W. Fiers, Nucleic Acids Res., 10, (1982), 2487-2501.

90. R. Derynck, P.W. Gray, E. Yelverton, D.W. Leung, H.M. Shepard, R.M. Lawn, A. Najarian, D. PeMica, F.E. Hagle, R.A. Hitzeman, P.J. Sherwood, A.D. Levinson, and D.V. Goeddel, in From Genes to Protein:

Translation into

Biotechnology, Ahmad, F., Schultz, J., Smith E.E., and Whelan, W.J., Eds. , Academic Press, New York, 1982, 249-262.

478

91. L. Mulcahy, M. K ~ M , B. Kelder, E. Rehberg, S. Pestka, and D.W. Stacey, Methods E n z p l . 119, (19861, 383-396. 92. E. Remaut, P. Stanssens, G. Simons, and W. Fiers, K d 119, (1980), 366-375. 93. M.A. Innis, and F. McCormic, in Methods in Enzymology, Vol. 119, Interferons, Part C, Colowick, S. and Pestka, S., Eds., Academic Press, New York, 1986, 397-403. 94. T. Staehelin, C. Stahli, D.S.

Hobbs, and S. Pestka, Meth. Enzymol.,

79,

(19811, 589-595. 95. M. Evinger, S. Maeda and S. Pestka, Recombinant human leukocyte interferon produced in bacteria has antiproliferative activity.

J. Biol. Chem. 256,

(1981), 2113-2114. 96. J.R. Ortaldo, A. Mason, E. Rehberg, J. Moschera, B. Kelder, S. Pestka, and R.B. Herberman, J. Biol. Chem. 258, (1983), 15011-15015. 97.

J.R. Ortaldo, A. Mantovani, D. Hobbs, M. Rubinstein, S. Pestka, and R.B. Hebberman, Int. J. Cancer 31, (1983), 285-289.

98. W.C. McGregor, and A.H. Ramel, In: Recombinant DNA Products: InsulinInterferon- Growth Horome, pp. 129-133, Bollon, A.P.

(ed.) Chemical Rubber

Co., Boca Raton, Florida, 1984.

99. J.U.

Gutterman, S. Fine, J.

Quesada, S.J.

Hornings, J.F.

Levine, R.

Alexanian, L. Berhardt, M. Kramer, H. Spiegel, W. Colburn, P. T r m , T. Merigan, and

2.

Dziewanowska, Ann. Intern. Med. 96, (1982), 549-556.

100. S.J. Tarnowski, Pharmac. Tech. 7, (1983), 70-79.

101.

P. Dunnill, Chem. Indust. 22, (19821, 877-879.

102.

J.W. Eveleigh, Affinity Chromatography and Related Techniques, pp. 293-303, Gribnau, T.C.J.,

Visser, J. and Nivard, R.J.F.

(eds) Elsevier, New York,

1982. 103. M. Maze, and G.M. Gray, Biochemistry 19, (1975), 2351-2358.

479

104. J.P. Chen, and H.M. Shurley, Res. 7, (1975), 425-434. 105. K.H. Brooks, and T.L. Feldbrush, J. Inununol. 127, (1981), 963-967. 106.

C.D.

Benjamin, A. Miller, E.E. Serarz, and M.A. Harvey, J. Ilnnunol. 125,

(1980), 1017-1025.

107. T. Staehelin, 9. Durrer, J. Schmidt, B. Takacs, J. Stocker, V. Miggiano, C. Stahli, M. Rubinstein, W.P. Levy, R. Hershberg, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 76, (1981), 1848-1852. 108. T. Staehelin, B. Durrer, J. Schmidt, 9. Takacs, J. Stocker, V. Miggiano, C. Stahli, H.-F. Kung, D.S. Hobbs, W.P. Levy, J.A. Moschera, and S. Pestka, Tex. Rep. Biol. Med. 41, (1982), 198-208. 109. N.G. Anderson, D.D. Willis, D.W. Holladay, J.E. Caton, J.W. Holleman, J.W. Eveleigh, J.E. Attrill, F.L. Ball, and N.L. Anderson, Analyt. Biochem., 66, (1975), 159-174. 110. S.J. Tarnowski, and R.A. Liptak, Jr. Adv. Biol. Processes 2, (1983), 271-287. 111. S.J. Tarnowski, S.K. Roy, R.A. Liptak, D.K. Lee, and R.Y. Ning, R.Y. Meth. En~ym01. 119, (1986), 153-165. 112. A.H. Nishikawa, and P. Bailon, J. Solid-Phase Biochem. I, (1986), 33-49. 113. S. Pestka, and S.J. Tarnowski, Pharmacology and Therapeutics 29, (19851, 299-319. 114. D.R. Thatcher, and Panayotatos Meth. Enzymol. 119, (19861, 166-177. 115.

S. Pestka, 9. Kelder, D.K.

Tarnowski, and S.J. Tarnowski, Analyt. Biochem.

132, (1983), 328-333. 116. G. Bodo, and I. Fogy, In:

the Biology of the Interferon System, pp. 23-27,

Kirchner, H. and Schellekens, H. (eds) Elsevier Science publishers B.V., Amsterdam, 1965. 117.

J.A. Langer, and S. Pestka, Meth. Enzymol. 119, (19861, 248-255.

480

118. S. Pestka, J.A.

Langer, K.C. Zoon, and C.E. Samuel, Am. Rev. Biochem. 56,

(19871, 727-777. 119. Y.K. Yip, R . H . L . Pang, C. Urban, and J. Vilcek, Proc. Natl. Acad. Sci. U.S.A. 78, (19811, 1601-1605. 120. Y.K.

Yip, B.S.

Barrowclough, C. Urban, and J. Vilcek, E d 79, (19821,

1820-1824. 121. Y.K. Yip, B . S . Barrowclough, C. Urban, and J. Vilcek, Science 215, (19821, 411-413. 122.

I.A. Braude, mth. E n z p l . 119, (19861, 193-199.

123.

E. Rinderknecht, B.H. O'Connor, and H. Rodriguez, J. Biol. Chem. 259, (19841, 6790-6797.

124. M.P. Langford, J.A. Georgiades, G.J. Stanton, F. Dianzani, and

H.M.

Johnson,

Infect. Inunun. 26, (19791, 36-41. 125. M. Dew, J. van Dame, H. Claeys, H. Weening, J.W. Heine, A. Billiau, C. Vermylen, and P. DeSomer, Eur. J. Innnunol. 10, (1980), 877-883. 126. S. Pestka,

8.

Kelder, P.C.

Familletti, J.A.

Moschera, R.

Crowl, and

E.S. Kempner, J. Biol. Chem. 228, (1983), 9706-9709. 127. S. Pestka, S. (ed.) Meth. hzymol. 119, (1986), 1-729. 128. S. Baron, F. Dianzani, and G.J. Stanton, Tex. Rep. Biol.

Med.

41, (19821,

1-410. 129. S. Baron, F. Dianzani, and G.J. Stanton, g d 41, (19821, 411-715.