Pichia Pastoris

8/17/2019 Pichia Pastoris

1/5

Biotechnology

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Extracellular Matrix

9 Vuorela, A., Myllyhaju, J. Nissi,

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Pihlajaniemi,T. and

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15 Fertala, A., Ho lmes , D.F., Kadler, K. E., Sieron, A. L. and

Prockop, D.J. ( 1996) J Biol. Chem. 27 I , 14864- I48 69 Received

28

February

2000

Expression of recombinant human type 1-111 collagens in the yeast Pichia pastoris

J. Myllyharju ,M.Nokelainen, A. Vuorela and K.

1

Kivirikko

Collagen Research Unit, Biocenter and Department of Medical B iochemistry, University

of

Oulu, P.0 Box 5000,

FIN-900

I 4 Oulu, Finland

Abstract

An efficient expression system for recombinant

human collagens will have numerous scientific

and medical applications. However, most recom-

binant systems are unsuitable for this purpose, as

they do not have sufficient prolyl 4-hydroxylase

activity. We have developed methods for pro-

ducing the three major fibril-forming human

collagens, types I, I1 and 111, in the methyl-

otroph ic yeast

Pichia

pastoris.

These methods are

based on co-expression of procollagen polypeptide

chains with the a and P-subunits of prolyl 4-

hydroxylase. T h e triple-helical type-I,

-11 and

-111 procollagens were found to accumulate pre-

dominantly within the endoplasmic reticulum of

the yeast cells and could be purified fro m the cell

lysates by a procedure that included a pepsin treat-

ment to convert the procollagens into collagens

and to digest most of the non-collagenous proteins.

All the purified recombinant collagens were ident-

ical in 4-hydroxyproline conten t with t he corres-

ponding non-recombinant human proteins, and

all the recombinant collagens formed native-type

fibrils. T h e expression levels using single-copy

integrants and a

2

litre bioreactor ranged from

0.2

to

0.6

g/l depending on the collagen type.

Key words: methylotrophic yeast, procollagen, prolyl 4-hy-

droxylase.

Abbreviation s used: aMF,a matingfactor; proa

I (I),

proa 11) and

proa

I (Ill) chains, proa I chains

of

type-I,

11

and -111 procollagen,

respectively; proa2(1) chain, proa2 chain of type-I procollagen.

To

whom correspondence should be addressed (e-mail

[email protected]).

Introduction

The collagen family consists of about

20

proteins

formally defined as collagens and more than 10

additional proteins with collagen-like domains

[l-31.

Type-I collagen is now used as a biomaterial

in numerous medical applications and as a delivery

system for various drugs [4-61. In addition, all

gelatins are made from collagens. Th e collagens

used in all these applications have been isolated

from animal tissues and are liable to cause allergic

reactions in some subjects and carry a risk of

disease-causing contaminants. Th e various colla-

gen types have different properties, and therefore

some of the other collagens might be more suitable

for certain applications than type I. However, it

has been difficult or impossible to isolate sufficient

quantities of the other collagens from animal

tissues. It is obvious, therefore, that an efficient

large-scale recombinant system for the production

of human collagens would have numerous ap-

plications in medicine.

Most recombinant systems now available for

large-scale production of proteins cannot be used

as such for the production of recombinant colla-

gens, as bacteria and yeast have no prolyl 4-

hydroxylase activity, and insect cells [7] and the

mammary gland [8] have insufficient levels of th is

enzyme activity. Prolyl 4-hydroxylase, an aJI

tetramer in vertebrates, plays a central role in the

synthesis of all collagens, as the 4-hydroxyproline

residues formed are essential for the folding of the

newly synthesized collagen polypeptide chains

into triple-helical collagen molecules [2 9 10].

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Biochemical Society Transactions (2000) Volume 28, part 4

Therefore, the recombinant collagen polypeptide

chains expressed in most systems will remain as a

non-triple-helical, non-functional protein or, if

the cells are grown at low temperatures, the chains

may form molecules with unstable triple helices.

We have demonstrated that co-expression of

polypeptide chains of various types of human

collagen with the two types of subunit of human

prolyl 4-hydroxylase can be used for efficient

recombinant expression of human collagens in

insect cells [7,11,12]. The recombinant type 1-111

collagens produced have been very similar if not

identical with the corresponding non-recombinant

proteins in their 4-hydroxyproline contents and

various other properties, and the highest ex-

pression levels obtained in suspension cultures

have ranged up to about 50mg/l [7,11,12]. In

addition, it has been demonstrated that this same

principle can be used for the high-level production

of an engineered form of human type-I collagen in

mouse milk [8]. We have recently applied the

principle to the high-level production of recom-

binant human type-1-111 collagens in the methyl-

otrophic yeast Pichia pastoris.

Expression of an active recombinant

human prolyl4-hydroxylase tet ram er

and the effect of co-expression with

collagen polypeptide chains

In order to study whether subunits of human

prolyl 4-hydroxylase are able to form an active

enzyme tetramer in yeast cells, cDNAs for the

human a and 8-subunits were cloned into the

Pichia expression vectors pARG815 (comple-

menting for arg4 in the host) and PA0815 (com-

plementing for his4 in the host), respectively, and

co-transformed into the GS200 (his4, arg4) P .

pastoris host train [13]. Initial attempts to express

an active human prolyl4-hydroxylase tetramer in

P. pastoris were only partially successful, as only a

minor fraction of the recombinant polypeptides

expressed were found in the form of the tetramer,

whereas the vast majority were present in un-

assembled forms [13]. A much higher tetramer

assembly level was obtained [13] when the signal

peptide of the /?-subunit was replaced by the

Saccharomyces cerevisiae a mating factor

(aMF)

pre-pro sequence by cloning the P-subunit c DNA

into the expression vector pPIC9 (generating

vector pPIC9p). This signal sequence gave the

highest amount of tetramer among the various

constructs studied, even though it also markedly

increased the secretion of the P-subunit into the

culture medium. Even in this P. pastoris strain,

however, the vast majority of the a andp-subunits

were found in unassembled forms.

T o study the expression of recombinant

human collagens in P. pastoris, cDNAs for the

proal chains of type-I, -11 and -111 procollagens

[proal(I), proal(I1 ) and proal (III)] were cloned

separately into the expression vector pPICZB and

transformed into a recombinant P. pastoris strain

expressing human prolyl 4-hydroxylase subunits

in which the 8-subunit had the S. cerevisiae a M F

pre-pro sequence ([13,14] and M. Nokelainen, A.

Vuorela, K. I. Kivirikko and J. Myllyharju, un-

published work). A highly unexpected finding was

that co-expression of the prolyl 4-hydroxylase

subunits with any of these procollagen polypep-

tide chains led to an up-to-about- 10-fold increase

in the amount of the enzyme tetramer with no

increase in the total amounts of its subunits

([13,14] and

M.

Nokelainen, A. Vuorela, K. I.

Kivirikko and J. Myllyharju, unpublished work).

Pulse-chase experiments indicated that the half-

lives of the recombinant enzyme tetramers ex-

pressed in

P.

pastoris without co-expression with

collagen polypeptide chains were only about

50 min [14], while co-expression with the proa-

l( II1) chains increased this half-life to 12.5 h and

co-expression with the p roal ( I) chains gave a half-

life of 6.5 h, i.e. 8 times that of the strain expressing

the enzyme alone but

50%

of that of the strain

co-expressing prolyl 4-hydroxylase with the

proal (I1 ) chains [141. The difference in half-life

between the strains co-expressing the proal (I )

and proal( II1) chains is likely to be related to the

level of procollagen expression, that of type-I

procollagen homotrimers being 35-70 yoof that of

type-I11 procollagen. T he data thus indicate that

collagen synthesis in P .pastoris, and probably also

in other cell types, involves a highly unusual

control mechanism, in that the production of a

stable prolyl 4-hydroxylase tetramer requires the

expression of collagen polypeptide chains, whereas

the production of collagen molecules with stable

triple helices requires the expression of active

prolyl 4-hydroxylase [13,141.

Expression of human type-I -11 and -111

collagens in shaker flasks

The strains described above were used to study

the expression of recombinant type-I , -11 and -111

procollagen homotrimers in P. pastoris. In order

to express type-I procollagen heterotrimers, a

cDNA for the proa2 chain of human type-I

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procollagen [proa2(1)] was cloned into the

pBLA DE IX vector (complementing for adel in

the host; M. Nokelainen, H. Tu, A. Vuorela,

H. Notbohm , K. I. Kivirikko and J. Myllyharju,

unpubl ished work). A stra in expressing prolyl 4-

hydroxylase was first generated to a yJC300 (his4,

arg4, adel)

P.

pastoris host strain by cloning a

cDNA for the a-subunit into the pBLARG IX

vector (complementing for arg4 in the host), and

this construct was co-transformed with the

pPIC9/3 expression vector (see above) into the

yJC300. This was followed by subsequent trans-

formations of the pPICZB vector containing the

proal(1) cDNA and pBLADE IX vector con-

taining th e proa2( I) cD NA.

All the P. pastoris strains expressing pro-

collagen were found

to

produce full-length proa

chains ([13,14] and M. Nokelainen, A. Vuorela,

K. I. Kivirikko and J . Myllyharju, unpublished

work). Th e p r o d (I) chains, when expressed

alone, and the proal(I1) and proal(II1) chains,

each formed triple-helical molecules with collagen

domains that were resistant

to

pepsin digestion,

whereas no pepsin-resistant chains were obtained

when the proa2(1) chains were expressed alone.

Co-expression of the proal(1 ) and proa2(I) chains

led to the formation of heterotrimeric type-I

procollagen molecules with the correct 2

:

1 chain

ratio (M. Nokelainen, H. Tu, A. Vuorela,

H.

Notbohm, K. I. Kivirikko and J. Myllyharju,

unpublished work). Studies by SDS/PAGE

under reducing and non-reducing conditions

indicated that all the proa chains and also the

a1 111) chains produced by pepsin digestion

from the corresponding procollagen molecules

formed disulphide-bonded trimers ([13] and M.

Nokelainen,

A

Vuorela,

K .

I. Kivirikko and J.

Myllyharju, unpublished work).

T h e thermal stability of the pepsin-resistant

recombinant collagens was studied using digestion

with a mixture of trypsin and chymotrypsin after

heating to various temperatures

[15].

T h e

T

values of the recombinant type 1-111 collagens

were approx. 38 C, which is 2-3

C

lower than

that found in vivo ([13] and J. Myllyharju, M.

Nokelainen, A. Vuorela and K.

I .

Kivirikko, un-

published work). Amino acid analysis

of

the

recombinant type-I I I collagen purified from

shaker-flask cul tures showed that the degree of 4-

hydroxylation

of

the proline residues was 44.2

yo

whereas the corresponding value for non-recom-

binant human type-I I I collagen was

5

1.6

[

131.

The best level

of

type-I11 collagen expression

obtained in shaker-flask cultures was approx.

15 mg/l, whereas the levels obtained for type-I

and -11 collagens were approx. 35-70

yo

of that of

type-I11 collagen ([13,14] and M. Nokelainen,

A. Vuorela, K. I. Kivirikko and J. Myllyharju,

unpublished work).

The triple-helical type-I,

-11

and

-111

pro-

collagen molecules produced in

P.

pastoris were

found to accumulate predominantly inside the

yeast cell, only about 10% being found in the

culture medium. This is surprising, as triple-

helical procollagen molecules are rapidly secreted

into th e extracellular space from various animal

cells. Replacement of the signal sequence of the

human proal(II1) chain with the

S.

cerevisiae

a M F pre-pro sequence led

to

only a slight im-

provement in secretion, and the total expression

level

of

type-I11 procollagen with the a M F

pre-pro sequence was lower than that with the

authentic signal peptide [16]. Immunoelectron

microscopy indicated that the recombinant pro-

collagen molecules accumulated within th e endo-

plasmic reticulum and did not proceed any further

in the secretory pathway [16]. Th e lackof secretion

may have been related to the large size

of

the

procollagen molecule.

Expression of human type-I -11 and -111

collagens in

a

bioreactor

Many previous studies have shown that shaker-

flask conditions are not optimal for protein pro-

duction in P. pastoris, du e to the lack of sufficient

0

and marked increases in expression levels are

usually obtained in bioreactors

[

17,181. As the

K

of

0

in the prolyl4-hydroxylase reaction is about

40 pM [19], the

0

concentration within the lumen

of the endoplasmic reticulum is also likely to be

rate-limiting for hydroxylation in shaker-flask

cultures. Thus it could be expected that the

differences in 4-hydroxyproline conten t between

the recombinant and non-recombinant collagens

may disappear when the recombinant collagens

are produced in a bioreactor. The type-I pro-

collagen homotrimers and heterotrimers and the

type-I1 and -111 procollagens were therefore

expressed in a 2 litre B. Braun Biostat

C

bioreactor

equipped with an

0

supply system, whereupon

their expression levels were indeed markedly

higher than in the shaker-flask cultures, ranging

from about 0.2

to 0.6

g/1 (M . Nokelainen,

H.

T u ,

A.

Vuorela, H. Notbohm, K. I. Kivirikko and J.

Myllyharju, unpublished work). It should be

noted that all the experiments reported in this

paper were carried out with single-copy integ-

rants. It has been demonstrated previously that

355 2000

Biochemical Society


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Biochemical Society Transactions (2000) Volume 28, part 4

the levels of expression of various proteins in

P.

pastoris increase markedly with the number of

DN A copies, at least up to

30 50

copies [17,20,21].

Th e present system can thus be optimized for the

production of very large amounts of various

collagens.

The recombinant collagens produced in the

bioreactor were purified by pepsin digestion and

selective salt precipitation followed by Sephacryl

S-500HR

gel filtration in the AKTA explorer

system (Amersham Pharmacia Biotech). All the

recombinant collagens produced were found to be

essentially pure when analysed by SDS/PAGE

followed by Coomassie Brilliant Blue staining

(Figure1).Amino acid analyses showed that the

4

hydroxyproline contents of all the purified recom-

binant collagens were identical with those reported

for the corresponding non-recombinant human

proteins (M. Nokelainen, A. Vuorela, K . I . Kivi-

Figure I

SDSlPAGE

analysis

of

purified recombinant human

collagens expressed in P.

pastoris

The long arrow indicates the a1 chains of the type-I collagen

homotrimer (lane I ) and heterotrimer lane2), and type-ll (lane3)

and type-Ill collagens (lane 4). The short amw indicates the a2

chain of the type-I collagen heterotrimer (lane

2).

1 2 3 4

Figure

2

N-termini

of

the a-chains of the purified recombinant

human collagens

The N elopeptide sequences are underlined. The amws indicate

the pepsin cleavage sites, while the N-terminal amino acid of the

a-chains of the final recombinant collagens is shown in bold.

J

a

1

I). . G N F ~ G G I S V P G P M G P S .

.

V W MGLM

...

2 I).. GNFMQYDGKG

a1@I ...NFM-GGAO hXMQGPMGPM ...

a1

@I ...Q

NYSPQYDSYDVKSGVAVGCL.AGYP

J

4

rikko and J. Myllyharju, unpublished work). N-

terminal sequencing of the polypeptide chains of

the recombinant collagens showed that in most

cases pepsin digestion had removed several resi-

dues from the N-terminus of the telopeptide

domain, but in the case of the

a2 I )

chain only one

residue had been removed and occasionally, if

pepsin digestion was incomplete, the

a2 I ) chains

had two additional N-terminal amino acids (i.e.

the cleavage had left the last two amino acids of the

N-propeptide on the N-terminus of the chain;

Figure 2). All the recombinant collagens produced

in P. pastoris were found to form native-type

fibrils (M. Nokelainen, H. Tu, A. Vuorela, H.

Notbohm, K.

I.

Kivirikko and J. Myllyharju,

unpublished work), which indicates that the dif-

ferences at the N-terminus do not influence the

fibrillar properties. This conclusion is supported

by a recent study on pepsin and pronase treatment

of rat non-recombinant type-I collagen molecules,

indicating that chains with shortened N-termini

form fibrils that are identical with those formed

from full-length chains [22]. It thus seems likely

that the recombinant procollagens produced in

P.

pastoris can be used for numerous applications

that currently require collagens purified from

animal tissues.

Wethank Dr.James Cregg, Keck Graduate nstitute of Applied Life

Sciences, for the gift of the P pastoris host strains and the

pBLARG IX and pBLADE IX vectors, and Raija Juntunen, Anne

Kokko, Eeva Lehtimaki, Minna Siunra and Tanja VaisLnen for their

expert technical assistance. This work was supported by grants

from the Health Sciences Council ofthe Academy of Finland, from

the European Commission B104-Cr96-0537),rom the National

Institutes of Health (ROI AR45879) and from FibroGen (South

San Francisco,CA, U.S.A.).

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Biochem. 80, 34 1-348

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36083-36088

Received I March

2000

Towards a fibrous compos ite with dynamically controlled stiffness

:

essons

from echinoderms

J. A. Tro t te r * , J. Tipper*, G. Lyons-Levy*, K.

Chino*,

A. H. Heuert . Z. L iu t ,

M.

Mrksichf,

C.

Hodnelandl,

W.

S.

Dillrnoref. T. J Koobtj, M. M. Koob-Ernundstj, K. Kadlery and D. Holrnesy

*Dep artment o f Cell Biology and Physiology, University of Ne w Mexico School

of

Medicine, Albuquerque,

NM 87 3 I, U.S.A., +Departme nt o f Materials Science and Engineering, Case Weste rn Reserve University,

I0900 Euclid Ave., Cleveland,

OH 44 106,

U.S.A.,

f

Department of Chemistry, Universrty of Chicago,

5735 S. Ellis Ave., Chicago, L 60637, U.S.A., SShrinen Hosp ital for Ch ildren, I2502 N. Pine Drive, Tampa,

FL 336 12, U.S.A., and flWellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences,

unive rsity of Manchester, Stopford Building, Ox for d Road, Manchester M I 3 9PT,

U.K.

Abstract

Sea urchins and sea cucumbers, like other echino-

derms, control the tensile properties of their

connective tissues by regulating stress transfer

between collagen fibrils. The collagen fibrils are

spindle-shaped and up to 1 mm long with a

constant aspect ratio of approx.

2000.

They are

organized into a tissue by an elastomeric network

of fibrillin microfibrils. Interactions between the

fibrils are regulated by soluble macromolecules

that are secreted by local, neurally controlled,

effector cells. We are characterizing the non-linear

viscoelastic properties of sea cucumber dermis

under different conditions, as well as the struc-

tures, molecules and molecular interactions that

determine its properties. In addition, we are

developing reagents that will bind covalently to

fibril surfaces and reversibly form cross-links with

other reagents, resulting in a chemically controlled

stress-transfer capacity. The information being

developed will lead to the design and construction

of a synthetic analogue composed of fibres in an

Key words : collagen, interfibril lar cross-links, fibrils.

To whom correspondence should be addressed (e-mail:

[email protected]).

elastomeric matrix that contains photo- or electro-

sensitive reagents that reversibly form interfib-

rillar cross-links.

Collagenous tissues

The structural materials of animals are, for the

most part, composites containing insoluble fibres

in a non-fibrous matrix. Familiar examples

of

such

materials include the tendons, ligaments and

dermis of mammals. Th e mechanical properties of

these fibrous composites are due largely to the

contributions of the protein collagen, which self-

assembles into long, thin fibrils that may be

millimetres in length and nanometres in diameter

[l] Collagen molecules (approx. 300 nm long x

1 5 nm in diameter) within the same fibril become

covalently cross-linked through enzymic action.

As a result

of

cross-linking, the fibrils possess

high tensile stiffness and strength (on the order

of GPa). I n most cases we

do

not know how long

the individual collagen fibrils are; nor do we

know how stress is transferred between them. We

do know, however, that the composition and

organization

of

the tissues is such as to make

effective use of the tensile properties of the fibrils.

In addition to collagen fibrils, connective tissues

357 2000

Biochemical Society

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