continuous production of somatomedin c with immobilized transformed yeast cells

Appl Microbiol Biotechnol (1988) 28:215-221 Applied Microbiology

Biotechnology © Springer-Verlag 1988

Continuous production of somatomedin C with immobilized transformed yeast cells

Koji Sode 1., Peter Brodelius 1, Franz Meussdoerffer 1.*, Klaus Mosbach 1'2, and Joachim F. Ernst 3.**

1 Institute of Biotechnology, ETH-H6nggerberg, CH-8093 Ztirich, Switzerland 2 Pure and Applied Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 O0 Lund, Sweden 3 Biogen SA, CH-1227 Geneva, Switzerland

Summary. Yeast cells producing the growth hormone somatomedin C (SMC) were constructed and applied in the immobilized form continuously for a period of over 10 days in a flow- through bioreactor. The construction of the MF~I-SMC fusion vector p336/l is given as well as the results of the influence of various nutrients effecting hormone production. Immobilization of the transformed yeast cells is described and their application in a continuous bioreactor system. This study demonstrates the feasibility of a long- term and high-level hormone production by immobilized transformed yeast. The SMC productivities of free cells in batch and immobilized cells under continuous conditions were 0.2--0.3 and 0.5--0.6 mg per g wet cells and day, respectively.

Introduction

A major advantage of recombinant DNA technology is the possibility to readily produce signifi- cant amounts of valuable polypeptides. However, product synthesis and recovery from recombinant bacteria is often complicated by inaccurate formation of eucaryotic gene products, precipitation or proteolysis. The yeast alpha-factor mediated secretion system uses fusions of protein coding sequences to the yeast mating pheromone alpha factor preprosequence which directs the gene

Present addresses: * Research Laboratory of Resources Utili- zation, Tokyo Institute of Technology, Nagatsuta-cho, Midori- ku, Yokohama, 227 Japan • * Henkel KGA, Abteilung Biotechnologie Z33, Henkelstrasse 67, D-4000 Dfisseldorf-Holthausen, FRG • ** Glaxo Institute of Molecular Biology, CH-1227 Geneva, Switzerland

Offprint requests to: P. Brodelius or K. Mosbach

product in the secretory pathway (Bitter et al. 1984; Brake et al. 1984; Ernst 1986). Thus, the advantage of yeast as eucaryotic host for which de- fined cultivation conditions have been established may be combined with the benefits of secretion, leading to direct product recovery from the medium. A relative simple product purification may be expected since only few other proteins are otherwise secreted by the yeast cells.

Immobilization of the yeast cells capable of secreting a recombinant DNA product could make such a system even more attractive. An immobilized yeast preparation permits continuous operation at high cell densities and high flow rates without the danger of a "wash out" of cells. This is particularly interesting for continuous culture of plasmid bearing yeast (Walmsley et al. 1983). Furthermore, the product may be directly recovered from the medium without the necessity to harvest cells. In addition a much restricted growth of the immobilized cells should take place within the beads, and therefore the danger of plasmid loss (i. e. accumulation of non-producing cells) by uneven segregation should also be dimin- ished (Zakian and Kupfer 1982). Immobilized yeast technology has developed rapidly from the first small scale experiments (Larsson and Mos- bach 1979) to large scale fermenters, several m 3 in size, especially for the production of ethanol (Na- gashima et al. 1984). A prerequisite for such a production system is a suitable immobilization method which includes mild polymerization conditions but results in a durable matrix. After evalu- ation of several cultivation parameters we produced somatomedin C (SMC) with the immobilized yeast system continuously for over 270 h. SMC (also called insulin-like growth factor 1) is the main mediator of the effects of growth hormone on somatic growth (Nilsson et al. 1986). The

216 K. Sode et al.: Immobilized transformed yeast

stimulating activity of SMC on bone and nerve growth have been described suggesting potential applications of SMC to promote wound healing, or overall body growth (Nilsson et al. 1986; Hans- son et al. 1986).

Materials and methods

Construction of a yeast strain secreting SMC. The yeast alpha factor gene MFal was isolated from a yeast genomic library (Nasmyth and Redd 1980) using an oligonucleotide probe corresponding to amino acids 97 to 102 of the precursor [(5') GTA CAT TGG TTG C / G C C G / A / T G G (3')] according to the published sequence of MFal (Kurjan and Herskowitz 1982).

The MFal gene on a 1.7 kb EcoRI fragment was subsequently subcloned into pUC-18. The resulting plasmid (p220/3) was mutagenized by primer mutagenesis to introduce a convenient BgllI site at the junction between secretion leader and the four alpha factor repeats:

oligomer (3') TA T I T TCT CTA GAA CTT CGA ACC (5') unchanged (3') TA TTT TCT CTC CGA CTT CGA ACC (5')

lys arg glu ala glu ala trp

The mutagenized vector was further modified by destroy- ing a B#III site 5' of the MFal promoter by filling-in the pro- truding ends with Klenow polymerase I and deoxynucleotide triphosphates, followed by ligation. The resulting plasmid was named p254 (Fig. 1).

The actual MFal/SMC fusion using modified MFal gene was made in two steps (Fig. 1): (1) insertion of a 500bp HindIII fragment carrying a synthetic SMC gene starting with an unique Ncol site (Buell et al. 1985) into the HindIII site of p254 (resulting plasmid F-9) and (2) cutting of plasmid F-9 with BgIII and Ncol, simultanous $1 treatment and religation. The correct fusion of the alpha-factor secretion leader (plasmid pS30/25) had the following sequence (glycine is the first amino acid of SMC):

(secretion Ieader)-AAA-AGA-GGT-CCA-(SMC) lys arg gly pro

The MFal/SMC fusion was introduced into a yeast shuttle vector carrying origins of replication for E. coli and yeast (ori; 2 ~ origin of replication), as well as selectable markers for both organisms (E. coli: bla; yeast: URA3).

The plasmid p336/1 (Fig. 1) was transformed into yeast strain BJ1991 (MAT ura3-521eu23,112 trpl prbl-l122 pep4-3) (E. Jones, Carnegie-Mellon University, unpublished results).

Cell cultivation. Transformed yeast cells were grown selectively in SD-medium (Sherman et al. 1981) containing yeast nitrogen base (1.7 g/l), ammonium sulfate (5 g/l), leucine (30 mg/l) and tryptophan (20 rag/l) at 30°C on a gyratory shaker (150 rpm) to an optical density (660 nm) of 2. These cultures were used to inoculate batch cultures in "production medium", consist- ing of SD-medium containing 4% casamino acids and tryptophan (inoculum was 10% of final volume of production medium) and for immobilization experiments.

Electrophoresis. SDS-PAGE was carried out according to Laemmli (1970). Medium (150 ~tl) was taken from the yeast cultivation in stationary phase and it was dialyzed against l0 mM Tris-HCl buffer, pH 7.2. Loading buffer was added

MF(t 1 / PUR

H

H N H

SMC

CYCl R

p160/1 10.5 kb

H I ARS1

U R A 3 ~

y 2gofi

MF~I SMC ~,~IL-.......~ H

ARS1

PUR I p336/1 11.3 kb

URA3 2~od

Fig. 1. Construction of the MFal-SMC fusion vector p336/1 as described in the Methods section. Restriction sites are: H = HindIII; R = EcoR1 ; N = NcoI. CYC1 is the terminator fragment of the CYCl-promotor (Ernst and Chan 1985); ARS1 is a chromosomal replicator (Botstein et al. 1979); PUR is the upstream region of the yeast PYK1 promoter (Dumont et al. 1987); URA3 is a yeast gene described by Botstein et al. (1979); the arrows indicate the location of the fl-lactamase gene (i. e. the bla-gene); A represents a deletion which leads to the fusion of the SMC gene with the a-factor segment (see Methods)

K. Sode et al.: Immobilized transformed yeast 217

and the sample was treated for 5 min at 95°C before it was loaded on a 18% gel. Standard SMC from E. coli (kindly sup- plied by A. Schmitz) was used as reference. Gels were stained by Coomassie Blue according to standard procedures.

Immobilization. Linear prepotymerized polyacrylamide hydrazide (PAAH) was prepared as described (Freeman and Aha- ronowitz 1981). Entrapment of yeast cells cultivated in SD- medium containing leucine and tryptophan was carried out in the following manner:

Cells (2.5 g fresh weight) were mixed with 5% (w/w) PAAH (15 ml), 3% (w/w) alginate (5 ml) and water (2.5 ml). This mixture was added dropwise to 0.1 M CaCI2 (100 ml) to form spherical beads. After 30 rain the beads were collected and treated with 0.25% glyoxal (50 ml) for 5 min. The beads were then washed with medium and used for the experiments.

The cell, alginate and gtyoxal concentrations were varied by making appropriate changes in the standard procedure.

Alginate was dissolved by incubating PAAH-beads in 0.1 M sodium phosphate buffer, pH 7.5, for 30 min on a gyratory shaker (150 rpm).

Operation of immobilized cell reactor. The conditions used for the continuous operation of the immobilized ceil reactor are indicated in the text and in the legends of Figs. 5--7.

Analytical procedures

SMC. SMC amounts within cells was determined as described (Ernst 1986). The concentration of SMC in media was determined with a commercially available radioimmuno assay (Ni- chols Institute, San Juan Capistrano, CA) according to the in- structions from the supplier.

Glucose. Glucose concentrations were determined after appropriate dilutions with a glucose analyzer (Yellow Springs In- struments).

Respiration. Cells (0.1 g fresh weight) or the corresponding amount of immobilized cells were collected by centrifugation and subsequently suspended in fresh growth medium (5.0 ml). The mixture was saturated with air and the respiration mea- sured at 25°C with an 02 electrode (Biometer, B. Braun Mel- sungen AG).

tO

-, ~O /

s 0

5 0.1

1 2 3 4 5

days

Fig. 2, Growth and secretion of SMC by the transformed yeast strain in batch culture in SD medium. ( - - O - - ) celt growth (OD66o); ( - - • - - ) SMC in medium; ( - I I - - ) SMC in cells

growth phase as illustrated in Fig. 2. The presence of SMC in the culture medium was shown by SDS-PAGE as illustrated in Fig. 3. The molecular weight of SMC is around 7000. Furthermore from the electrophoresis it is evident that SMC is the major extracellular protein in stationary phase. SMC present in the culture medium was purified and partly characterized. The amino and carboxyl terminal amino acids were identical to human

1 2

Results

Construction o f a yeast strain secretin 9 S M C

The fusion of the SMC coding sequence to the yeast alpha factor preprosequence was accom- plished in two steps as outlined in Fig. 1. In the first step the M F a l gene was isolated and appro- priately modified and in the second step this modified gene was fused to the SMC sequence.

It was verified that produced SMC is secreted by the transformed yeast and that the secreted SMC was not altered during secretion. SMC accu- mulated in the medium during batch culture of free cells after the cells entered late logarithmic

Fig. 3. SDS-PAGE of SMC as described in Materials and methods. (1) Yeast culture medium taken in stationary phase; (2) purified SMC from E. coli. The arrows indicate the band corresponding to SMC


SMC (A. Schmitz, unpublished results). The biological activity of the secreted SMC was identical to authentic SMC as determined according to Ernst (1986). The SMC productivity of free cells under batch conditions was 0.2--0.3 mg per g wet cells and day.

Immobilization of the transformed yeast

The transformed yeast cells were immobilized by entrapment in polyacrylamide hydrazide (PAAH) cross-linked with glyoxal (Freeman and Aharono- witz 1981). Attempts to make spherical beads (having good flow properties) by dispersing the cell/polymer mixture in an inert hydrophobic phase (Nilsson et al. 1983) prior to cross-linking failed. However, spherical beads were obtained by adding a low concentration of sodium alginate to the cell/PAAH suspension and by subsequently adding this mixture dropwise to a medium containing calcium ions. The beads were treated with an appropriate concentration of glyoxal to cross-link PAAH.

We investigated in the following the impact of immobilization parameters on viability (reflected by their respiration) of the immobilized yeast cells. In particular, variations in the glyoxal concentration, which determines the degree of cross- linking and of the alginate concentration, which defines the pore dimensions, have been tested. Neither the glyoxal (0.1%--1.0% w/w) nor the alginate concentration (0.3%--0.9% w/w) used during immobilization have any decisive effect on cell respiration as summarized in Table 1. When alginate was dissolved by washing the beads with phosphate buffer a somewhat reduced cell respi-

Table 1. Effect of immobilization parameters on respiration of SMC producing yeast. Beads (5 g) (initial cell concentration 10% w/w) were incubated in SD medium (50 ml) at 30°C for 40 h

Prepa- PAAH Alginate Glyoxal Phosphate Relative ration (%) (%) (%) treatment respi- number ration

(%)

1 0.0 1.0 0 . 0 - 100 2 3.0 0.6 0.25 - 93 3 3.0 0.6 0.10 + 64 4 3.0 0.6 0.25 + 62 5 3.0 0.6 0.50 + 63 6 3.0 0.6 1.0 + 47 7 3.0 0.3 0.25 + 60 8 3.0 0.9 0.25 + 55

200

100

0

i

o~

t -

O .m

> .m

incubation time (h) Fig. 4. Relative respiration of immobilized cell preparations as function of incubation time. The samples studied correspond to preparations number 2 ( - - © - - ) , number 4 ( - - [ ] - - ) and number 6 ( - - zx - - ) in Table 1. The initial respiration of preparation 2 was taken as 100% relative respiration. 5 g beads (initial cell concentration 10% w/w) were incubated in SD medium (50 ml) at 30°C on a gyratory shaker (150 rpm)

ration was initially observed. Full respiration was restored after a few hours incubation in medium as illustrated in Fig. 4. In further experiments the alginate was not removed.

Impact of medium composition on SMC production

The impact of different carbon sources on SMC production by immobilized yeast was first evalu- ated under batch conditions. Somatomedin C levels were higher on a rich carbon source (i. e. glucose) than on poor substrates like glutamate or casamino acids as summarized in Table 2. How- ever, the former also favored growth and subsequent slight leakage of ceils out of the beads. Sim- ilar SMC secretion rates were observed at 20 and 37 ° C.

In additional experiments under continuous culture conditions beads (5 g) containing yeast cells (1 g wet weight) were perfused (0.18 ml/min) in a column reactor (11 ml working volume) with SD medium containing tryptophan, leucine and 2% glucose (medium selective for plasmid main- tenance). When steady state had been established (80 h), glucose was replaced by casamino acids (2%) as carbon source. A rapid drop in SMC concentration took place after the shift and after an operation for 40 h with medium containing casamino acid no SMC was found in the effluent.

Table 2. Impact of the medium composition on SMC production by immobilized yeast cells under batch conditions. Beads (5 g) containing cells (10% w/w) were incubated in SD medium (30 ml) at 30°C on a gyratory shaker at 150 rpm containing various additions

Addit ion Incu- SMC Cell bation concen- density time tration ( 0 D 6 6 0 )

(h) (mg/1) O

Glucose (20 g/l) 40 1.24 0.21 Glucose (20 g / l ) +

casamino acids (20 g/l) 40 1.74 0.27 Glucose (20 g / l ) +

casamino acids (20 g/l) 60 2.23 0.97 Galactose (20 g/l) 40 0.28 0.19 Glutamate (20 raM) 40 0.15 0.02 Glutamate (20 m M ) +

casamino acids (20 g/ l ) 40 0.20 0.02 Casamino acids (20 g/l) 40 0.25 0.02

~ I ¸

.6 f 0

1 . 0 'T

~.8

• -~ .6

~.4

0


20

E

10

10 20 4'0 6'0 8'0 100

glucose in medium (g I "I)

Fig. 5. Effect of glucose concentration on the performance of a continuous reactor containing immobilized transformed yeast. ( - - • ) SMC concentration; ( - - • - - ) glucose concentration in effluent; ( - - • - - ) OD660. 4 g beads (initial cell concentration 25% w/w) were used in a column reactor (working volume 11 ml) at a dilution rate of 0.98 h-~. Measurements were performed at steady state after 80 h continuous operation with SD medium

We determined the glucose concentration yielding optimal SMC production while restrict- ing maximally the growth in batch and under continuous culture conditions. Under batch conditions SMC production was highest at a low glucose concentration (20 g/l) as indicated in Table 3. In a continuous operation maximum SMC production was observed at a considerably higher glucose concentration (50 g/l) as shown in Figure 5. Steady state (constant glucose and SMC levels) was reached after 50 hours operation independent of glucose concentration. At a low glucose concentration (20 g/l) cells grew out of the beads as indicated by an increase in medium OD66o, while at higher glucose concentrations ( > 5 0 g/l) no leakage of cells was observed (Fig. 5). Therefore, all subsequent experiments were performed at glucose concentrations of 50 g/l.

Table 3. Effect of initial glucose concentration on SMC production by immobilized yeast cells. Beads (4g) containing cells (8% w/w) were incubated for 80 h in SD medium (50 ml) containing different concentration of glucose

Initial SMC Final glucose concentration glucose concentration (rag/l) concentration (g/l) (g/l)

20 0.65 0 50 0.10 15

100 0.12 30 150 0.08 35

Optimization of the continuous process

In order to achieve optimal continuous SMC production with the immobilized yeast, the inter- dependence of glucose consumption, dilution rate and SMC production was determined. Measure- ments were performed after 80 h continuous operation of a small column reactor. As shown in Fig. 6 the highest SMC concentration is obtained at a dilution rate of about 1 ( h - ]). However, max- imal productivity is reached at a dilution rate of around 2 (h - ]) since the higher flow rate compen-

1.0- ~ 2

C J) 12~ & &

E ._O >,

= £ O O Q-

O o

r.,9 o- o

" . , - - % ~ . : : . . . . _,~

" '"- ,o

i i i

0 1 2 3 4 di lut ion rate (h -1)

'T

c- O

E 50

E 0

0 0

O

5

Fig. 6. Effect of dilution rate on the performance of a continuous reactor containing immobilized transformed yeast. ( - - [ ] - - [ ] - - ) SMC concentration; (- © • . . O • .) SMC productivity; ( - - A -- - - A -- ) glucose consumption. 4 g beads (initial cell concentration 30% w/w) were used in a column reactor (working volume 11 ml). Measurements were performed at steady state after 80 h continuous operation with SD medium. Solid symbols represent the results of an independent experi- ment after 7 days continuous operation


1.0

m .8

,5

G) =o .4 o

.- 2o g 1-1 -'-l,q 50 g 1-1 ~'1

.2

I

O0 100 200 300

time (h)

Fig. 7. SMC production of an immobilized yeast cell reactor as function of incubation time. 10 g beads (initial cell concentration 8.4% w/w) were continuously perfused with a SD medium containing glucose as indicated in a column reactor (working volume 15 ml) at a dilution rate of 2.2 h -1

sates for the lower yield. In an independent ex- periment the same SMC concentration and productivity was observed after 7 days of continuous operation at a dilution rate of 2 (h -1) (Fig. 6, solid symbols). The glucose consumption increased steadily with the dilution rate up to a dilution rate of around 2 (h-~) and remained constant at higher dilution rates (Fig. 6).

Continuous production of SMC

A continuous production of SMC by immobilized yeast cells over an extended period of time is illustrated in Fig. 7. Increasing the glucose concentration from 20 to 50 g/l results in an increased production of SMC from around 0.6 to 0.8 mg/1 (cf. Fig. 5). This corresponds to a productivity of 0.5--0.6 mg SMC per g wet cells and day.

Discussion

The utilization of immobilized transformed cells for the production of high value compounds has only been studied to a limited extent. For in- stance, cells of Bacillus subtilis carrying plasmids encoding rat proinsulin entrapped in agarose beads produced proinsulin for extended periods of time (Mosbach et al. 1983). A major advantage of a process involving an immobilized biocatalyst is a continuous operation. However, for such a process the product should be extracellular. Here the alpha factor mediated secretion system of

yeast offers an applicable system (Bitter et al. 1984; Brake et al. 1984).

There are numerous procedures available for the immobilization of whole cells with retained viability (Mosbach 1987). Here we have used sodium alginate (Kierstan and Bucke 1977) in com- bination with polyacrylamide hydrazide (Freeman and Aharonowitz 1981) to make spherical beads of a durable matrix with good flow properties. The entrapped cells show a high survival rate. A similar procedure has been described for the immobilization of phenol degrading Pseudomonas sp. (Bettmann and Rehm 1984).

In batch culture the transformed yeast cells produced SMC in late logarithmic growth phase and after 3 days incubation the SMC concentration was around 12 mg/1 which corresponds to a SMC productivity of 0.2 to 0.3 mg per g fresh wet of cells per day. The SMC produced is secreted into the medium where it is stable for at least a few days (cf. Fig. 2). Furthermore, only a very limited number of proteins can be found in the medium (cf. Fig. 3) and therefore the purification of SMC produced by the yeast system should be re- latively simple.

The effects of various process parameters on the production of SMC by the immobilized yeast cells under continuous operation have been investigated. The low somatomedin C concentrations found when poor carbon sources were used could be a direct result of the carbon source itself or a mere consequence of the fact that under these conditions (no growth) the cell number in the beads was presumably much lower than on rich carbon sources where some growth occurred. However, upon substituting a rich medium (glucose) at steady state for a poor medium (casamino acids) resulted in a rapid decline in SMC concentration in the effluent. Furthermore, we established that under continuous operation higher glucose concentrations in the medium resulted in high SMC productivity and at the same time no cell leakage. A possible explanation for the reduced growth of cells at high glucose concentration may be the rapid formation of ethanol which inhibits the growth of the cell. Anyhow, at a high glucose concentration a continuous process may be operated over an extended period of time with essentially constant SMC productivity (cf. Fig. 7). A comparison of free cells in batch (Fig. 2) and immobilized cells under continuous operation (Fig. 7) show that SMC-productivities of 0.2--0.3 and around 0.5--0.6 mg per g wet cells and day, respectively, may be reached. The productivity of the immobilized system is thus twice


that of the batch fermentation system. In addition to this the immobilized cells may be used for an extended period of time (up to 10 days as illustrated in Fig. 7) resulting in a considerably higher total productivity for the immobilized cells.

We have reported here the utilization of immobilized transformed yeast cells for the continuous production of a recombinant DNA product (somatomedin C). The yeast has been transformed with a plasmid carrying a fusion of the SMC coding sequence to the alpha factor preprosequence of yeast. This is the first example of immobilized transformed yeast cells producing an extracellular foreign protein in high amounts under continuous conditions. The alpha factor mediated secretion system should prove valuable for the develop- ment of other continuous immobilized yeast cell processes for the production of various proteins.

Acknowledgements. This study has been supported in part by a grant from Biogen SA. Mr. K. Sode has been the recipient of a Swiss-Japanese exchange scholarship from Swiss Federal In- stitute of Technology.

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Received August 14, 1987/Accepted December 8, 1987

continuous production of somatomedin c with immobilized transformed yeast cells

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