integrated production of human insulin and its c-peptide

ELSEVIER Journal of Biotechnology 48 (1996) 241-250

Integrated production of human insulin and its C-peptide

Joakim Nilsson, Per Jonasson, Elisabet Samuelsson, Stefan Stghl*, Mathias UhlCn

Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden

Received 22 December 1995; accepted 1 March 1996

Abstract

The potential for the development of an integrated process for production of human insulin and its C-peptide in Escherichiu coli has been investigated. Human proinsulin was produced intracellularly in E. coli fused to two synthetic IgG-binding domains (ZZ) derived from staphylococcal protein A. High expression levels (3 g/l culture) of the gene product, which accumulated as inclusion bodies, was obtained. Solubilization of inclusion bodies by oxidative sulfitolysis and subsequent renaturation was performed directly after cell lysis and pellet wash. IgG affinity chromatography was used for efficient recovery of pure proinsulin fusion protein in a single step. Monomers of the proinsulin fusion protein constituted - 70%. A single step conversion of the fusion protein into insulin and C-peptide by trypsin and carboxypeptidase B treatment was achieved by engineering the junction between proinsulin and its affinity handle, ZZ. Characterization of the cleavage products by reversed phase chromatography (RPC) verified that human insulin and C-peptide were generated and that the ZZ affinity handle was resistant to cleavage. Human insulin and C-peptide were recovered with high yields by preparative reversed-phase high performance liquid chromatography (RP-HPLC). The potential use of the presented scheme for large-scale production of recombinant insulin and/or its C-peptide is discussed.

Keywords: Fusion protein; Proinsulin; Staphylococcal protein A; Affinity chromatography; Renaturation; Trypsin cleavage

1. Introduction

Human insulin produced in Escherichiu coli was

launched on the market in 1982 (Johnson, 1983),

but efforts to develop more cost-efficient produc-

tion schemes have not diminished. One recently

emerging reason for the renewed interest in proin-

sulin production is that several studies indicate

that the C-peptide, connecting the B- and A-

chains in proinsulin, has a clinical relevance also

* Corresponding author. Tel.: + 46 8 7908758; fax: + 46 8

245452.

(Johansson et al., 1992, 1993). In patients with

type 1 diabetes, who lack endogenous C-peptide,

0168-1656/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PII SO168-1656(96)01514-3

242 J. Nilwon rl rrl. /I Journd of Bmirc~hrzolog~ 48 (1996) 241 -250

administration of the peptide improves renal function, stimulates muscle and glucose utilization and improves blood-retinal barrier function (Jo- hansson et al., 1992, 1993).

An important part of modern biotechnology is to develop simplified production schemes for recombinant proteins by the integration of unit operations (Datar et al., 1993). Genetic product

design can be used in several ways to implement such schemes, i.e. by influencing the yield and localization of the product as well as to adapt the

gene product to specific unit operations suitable for large-scale downstream processing. The isoelectric point or the partitioning properties of a gene product can be altered by site-specific changes to allow ion exchange chromatography or two-phase extraction as primary purification

steps in the separation process (Dalbsge et al.. 1987; Kohler et al., 1991). Secretion strategies have been particularly interesting in the aspect of

process integration, since a large degree of the purification is already achieved by the product secretion (Blight et al., 1994). Expanded bed adsorption was recently combined with the secretion strategy for efficient recovery of a recombinant fusion protein, directly from a crude process mixture without prior cell removal (Hansson et al., 1994).

Early experiments suggested that secreted proinsulin should be more stable than intracellularly produced proinsulin (Talmadge and Gilbert, 1982) and consequently, significant efforts have been made to develop efficient secretion systems for human proinsulin (Chan et al., 1981; Stahl and Christiansen, 1988; Murby et al., 1991; Kang and Yoon, 1994). Nevertheless, secreted proinsulin as well as soluble cytoplasmic proinsulin have been demonstrated to be sensitive to proteolysis (Murby et al., 1991; Shen, 1984) and different gene fusion approaches have been investigated to stabilize the gene product (Murby et al., 1991; Shen, 1984). However, intracellular overexpres- sion of proinsulin, leading to aggregation, has proven to have a dramatically stabilizing effect on proteolysis (Shen, 1984; Guo et al., 1984). High expression levels, up to 30% of total E. cob cell protein, have been reported using this production route (Williams et al., 1982; Guo et al., 1984). The

inclusion body strategy for production of recombinant proteins, having the main advantages that the gene product normally is protected from proteolysis and the high expression levels, has be- come a more attractive alternative due to recent advances in large-scale in vitro renaturation of

recombinant proteins from intracellular precipi- tates (Rudolph, 1995).

Fusion of a target gene to a gene encoding a fusion partner with several useful features can be a rational way to enable the design of cost-efficient production schemes. Increased expression levels (Murby et al., 1994) product stabilization (Shen, 1984; Murby et al., 1991) and efficient

purification by affinity chromatography (Nygren et al., 1994) are some of the advantages that can be achieved by gene fusion strategies. In addition, the solubility of a recombinant protein during in vitro refolding can be increased more than lOO- fold with the use of a suitable fusion partner (Samuelsson et al., 1994). However, gene fusions introduce a problem into the downstream processing scheme, due to the fact that the fusion tail has to be cleaved off and removed. A number of chemical and enzymatic methods have been investigated for this purpose (Carter, 1990; Forsberg et al., 1992; Uhlen et al., 1992) and some of the more specific enzymes, having a substrate cleavage site containing four or more amino acids, e.g. H64A subtilisin, Factor Xa and IgA protease, are considered attractive (Nygren et al., 1994) with

the main drawback of being expensive. Trypsin, which cleaves C-terminally of basic amino acids, is inexpensive and readily available and has been used extensively to digest proteins to small peptides, but has found limited use in the processing of fusion proteins (deGeus et al., 1987; Carter. 1990). This is probably due to the expected low specificity of the enzyme, since most proteins contain numerous potential trypsin-sensitive sites. Nevertheless, proinsulin and also other recombinant proteins have been demonstrated to be spe- cifically cleaved at desired positions after refolding of the gene products (Kemmler et al., 1971; Frank et al., 1981; Cousens et al., 1987; deGeus et al., 1987; Wang et al., 1989), suggesting that a one-step conversion of a fusion protein containing proinsulin could potentially yield hu-

J. Nilsson et al. / Journal of Biotechnology 48 (1996) 241-250 243

man insulin and C-peptide with simultaneous removal of the fusion tail.

Here, we present an investigation where careful design of the gene product enables integration of unit operations, thus allowing the design of a straightforward process scheme for the production of a fusion protein which is cleaved in a single-step procedure to yield both native human insulin and its C-peptide.

2. Materials and methods

2.1. Production strain and plasmid

The E. cofi strain 017 (Olsson and Isaksson, 1979) was used as host for the plasmid pTrpZZ- R-proinsulin (Jonasson et al., 1996). This plasmid encodes a fusion protein, consisting of an IgG- binding ZZ affinity handle fused to human proinsulin with an arginine residue engineered between ZZ and proinsulin. In addition, the plasmid car- ries the gene conferring kanamycin resistance. Transcription initiation is under control of the E. coli trp promoter.

2.2. Cultivation and recovery of inclusion bodies

E. coli cells harbouring the plasmid pTrpZZ-R- proinsulin were grown overnight at 37°C in 200 ml Tryptic Soy Broth (Sigma, USA) containing 50 mg/l kanamycin monosulfate (Sigma). This starter culture (A,,, nm x 4) was used to inoculate a 3-l (working volume 2 1) bioreactor (Belach 3 1 bioreactor, type FLC3-A, Belach, Sweden). The medium consisted of 10 ml/l 87% glycerol, 2.5 g/l

(NH,)&&, 3 g/l KH,PG,.H,O, 2 g/l K,HP0,.2H20, 0.5 g/l Na,-citrate, 1 g/l yeast extract (Difco, USA), 1 g/l MgS0,.7H,O, 50 mg/l kanamycin monosulfate, 70 mg/l thiamine, 1 ml/l trace element solution (Jansson et al., 1996), and 0.65 ml/l vitamin solution (Jansson et al., 1996). Parameters controlled during cultivation were; pH, stirrer speed, temperature, oxygen saturation, glycerol feed and aeration rate. Foam was controlled by adding antifoam agent (Adecanol LG- 109). The pH was maintained at 7.0 with NH, (25%) and H,PO, (30%) feeding and the tempera-

ture was maintained at 37°C. The growth was controlled by glycerol-feeding to maintain a dis- solved oxygen tension of 30%. After 28 h cultivation (A,, nm = 180), fi-indole acrylic acid was added to a final concentration of 25 mg/l. Three h later, the cells were harvested (Aem nm = 210) by centrifugation at 5000 x g for 15 min. The cell- pellets were resuspended in washing buffer (50 mM Tris-HCl pH 8.0, 0.2 M NaCl, 0.05% Tween 20 and 1 mM EDTA) and stored at - 20°C until further use. For dry mass analysis, three 5-ml samples were withdrawn in preweighed glass test tubes from the harvested cell-culture. Cells were pelleted and washed once in distilled water and dried overnight at 110°C before determination of the mass. A cell pellet corresponding to 150 ml cultivation was thawed and disintegrated in a high pressure homogenizer (French press FA-073, Am- into, USA). The disintegrate, containing inclusion bodies, was pelleted at 15 000 x g for 30 min and subsequently washed 2 times, by resuspending in washing buffer followed by centrifugation, and then once with 50 mM Tri-HCl, pH 7.5. Washed inclusion bodies were stored at - 20°C.

2.3. Solubilization and renaturation

Washed inclusion bodies, containing ZZ-R- proinsulin, were solubilized and converted to its hexasulfonate derivative by oxidative sulfitolysis (Cole, 1967), essentially as described by Frank et al. (1981), by adding a solution containing 8 M urea, 0.8 M sodium sulfite (Na,SO,) and 0.3 M sodium tetrathionate (Na,S,0,.2H,O) (Cousens et al., 1987) to a final ZZ-R-proinsulin-concentration of - 2 mg/ml. The mixture was incubated at 37°C under gentle stirring for 6 h. After dilution 1: 1.5 with distilled water and subsequent centrifugation at 30000 x g for 20 min, the supernatant was dialysed 4 times (Spectra/Par 1 membrane, MWCO 6-8000, Spectrum Medical Industries Inc., USA) against 5 1 of 10 mM Tris-HCl, pH 8. Dialysed protein solution was chilled on ice and supplemented with 1 M glycine-NaOH, pH 10.5, to a final concentration of 0.1 M glycine-NaOH. In addition, /?-mercaptoethanol was added ( N 18 mol/mol fusion protein) (Frank et al., 1981). This reaction mixture was incubated at 4°C under gen-

244 J. Nilsson et al. I Journal of Biotechnology 48 (1996) 241-250

tle stirring for 20 h in a final volume of 450 ml, at a concentration of ZZ-R-proinsulin of - 0.8

mg/ml.

2.4. Affinity purljication and enzymatic cleavage

of the proinsulin fusion protein

After pH adjustment to 8 with acetic acid and centrifugation at 30000 x g for 20 min, the supernatant was subjected to IgG-Sepharose affinity chromatography (Moks et al., 1987) on a HR 16/10 column containing 12 ml IgG-Sep- harose@ (Pharmacia Biotech, Sweden) equilibrated with washing buffer, using an FPLC” system (Pharmacia Biotech). Aliquots of 50 ml were loaded at a flow rate of 3 ml/min. The column was washed with 3 bed volumes of washing buffer followed by 3 bed volumes of 10 mM NH,Ac, pH 8.0. Elution of ZZ-R-proinsulin was performed using 0.3 M HAc, pH 3.1. The eluate (160 ml) was monitored at 280 nm

and the fractions of interest were pooled. The flow rate during both washing and elution were 3 ml/min. Adjustment of pH to 8.0 was carried out by addition of 25% ammonia solution. The volume was reduced 5 times by ultrafiltration (Ultrasart Cell 50, MWCO 10000, Sartorius AG, Germany). Tween 20 was added to 0.1% before site-specific cleavage and conversion of proinsulin into insulin and C-peptide (Kemmler et al., 1971) by trypsin (T-2395, Sigma, USA) and car-

boxypeptidase B (Boehringer Mannheim, Ger- many) at a ZZ-R-proinsulin concentration of 12 mg/ml. The ratio of ZZ-R-proinsulin to trypsin was 1OOO:l (by mass) and the ratio of ZZ-R- proinsulin to carboxypeptidase B was 2OOO:l (by mass). The digestion was stopped after 30 min by the addition of trifluoroacetic acid (TFA) to pH 3. Acetonitrile (ACN) was added to 20%. The cleavage solution was stored at 4°C.

2.5. Preparative RP-HPLC

Aliquots (5 ml) of the cleavage solution were loaded on a reversed phase Kromasil C-8- column (particle size, 10 pm; diameter, 25 mm: length, 250 mm; Kema Nord, Sweden), at a flow rate of 5 ml/min at 30°C. The column was

previously equilibrated with 30% ACN, contain-

ing 0.25% pentafluoropropionic acid (PFPA). Elution was performed with a gradient of 30- 50% ACN, containing 0.25% PFPA, at 5 ml/min flow rate during 30 min. The absorbance was monitored at 220 nm. Relevant fractions were collected and stored at 4°C.

-3.6. Protein unalysis

SDS-PAGE was performed on a homoge- neous 12”/1) gel (BioRad Inc., USA) under reducing conditions, according to Laemmli (1970). Coomassie Brilliant Blue R-350 (Pharmacia Bio- tech) was used for staining. The homogeneity of IgG-affinity purified ZZ-R-proinsulin was analyzed by size exclusion chromatography on a Superdex 75 PC 3.2/10 column (Pharmacia Bio- tech), using the SMARTTM system (Pharmacia Biotech). The buffer was 200 mM sodium phos- phate pH 7.2, containing 15% ACN. The flow rate was 100 pl/min and the absorbance was measured at 214 nm. Cleavage products were analyzed by RPC on a pRPC C2/C18 SC 2.1/10 column (Pharmacia Biotech) using the SMARTTM system. Elution was performed using

a gradient of 31-36% ACN, containing 0.1% TFA, during 12 min at 25°C. The flow rate was 100 pl/min and the absorbance was measured at 214 nm. To determine the IgG-binding compo- nents after cleavage, IgG-Sepharose was added and incubated for 2 min during gentle mixing. The IgG-Sepharose was sedimented by centrifugation and the supernatant was analyzed by RPC on the SMARTTM system as described above. Insulin and C-peptide containing fractions from the preparative RP-HPLC step were analyzed by RPC as described above, using a gradient of 31-39% during 15 min for insulin- containing fractions and a gradient of 30-34% during 20 min for C-peptide-containing fractions, respectively. Internal standards, human insulin (Hoechst AG, Germany) and C-peptide fragment 3-33 (Sigma, USA) were used to iden- tify insulin and C-peptide, respectively. Relevant fractions were pooled and lyophilized, taken up in ultrapure water (MilliQ-Plus System, Mil- lipore, USA) and lyophilized a second time.


2.7. Protein quantljication

The amount of ZZ-R-proinsulin after IgG- affinity purification was calculated by absorbance measurement at 280 nm using the absorption coefficient 0.34 cm*/mg (Jonasson et al., 1996). Quantification of ZZ-R-proinsulin in harvested cells and before renaturation was performed by SDS-PAGE analysis on the PhastSystemTM (Phar- macia Biotech) using 8-25% polyacrylamide gradient gels and comparison to preweighed, IgG-affinity purified ZZ-R-proinsulin samples. The yields of insulin and C-peptide in the cleavage solution were calculated by integration of insulin and C-peptide peaks, separated by analytical RPC as described above, followed by comparison with a standard curve made by injection of known amounts of human insulin and C-peptide standards, respectively. The amounts of insulin and C-peptide, recovered by preparative RP-HPLC, were determined by measuring the mass of lyophilized insulin and C-peptide on an analytical balance.

3. Results

3.1. Product design and process set-up

The rationale behind our production scheme for human insulin and its C-peptide was to produce a fusion protein containing proinsulin, and to utilize an affinity handle with several advanta- geous features, and subsequently process the fusion protein in a single step to enable recovery of the final products. The ZZ tail derived from staphylococcal protein A (SPA; Nilsson et al., 1987) was selected as fusion tail since, (i) ZZ fusion proteins can be produced at high expression levels in E. coli (Hansson et al., 1994; Moks et al., 1987; Josephson and Bishop, 1988), (ii) ZZ-fusions allow efficient recovery by IgG-affinity chromatography (Moks et al., 1987) also after solubilization of inclusion bodies (Murby et al., 1994) indicating that ZZ efficiently adopts its structure during renaturation and (iii) the solubilizing properties of ZZ enable in vitro product refolding at high protein concentrations (Sa-

muelsson et al., 1991; Samuelsson et al., 1994). Furthermore, the ZZ-tag is rather small (15 kDa), contains no cysteine residues that could cause unwanted disulfide bridges and has been shown to be highly resistant to proteolysis (Moks et al.,

A c 1 + z 1 z tR+-tR-R+-tK--Ra

- w M *

IgG-binding proinsulin

1 Isolation of inclusion bodies 1

+

1 Solubilization and renaturation 1

1 IgG affinity chromatography 1

Site-specific cleavage

1 Reversed phase chromatographyl

J\

I C

Fig. 1. Schematic presentation of the ZZ-R-proinsulin fusion protein (25 kDa) and the investigated production scheme for recovery of human insulin and its C-peptide. (A) A trypsin- sensitive cleavage linker, comprising a single arginine residue was engineered between the ZZ fusion partner and proinsulin. The trypsin-sensitive processing sites flanking the C-peptide of proinsulin are also indicated. Arrows indicate expected trypsin cleavage sites. (B) The presented scheme for the production of human insulin and its C-peptide.

246 J. Nilsson et (11. I Journal of Biotechno1og.v 48 (1996) 241-250

A

1234M

I‘; 0.0

5.0 10.0 15.0

Time (min)

Fig. 2. Analyses of ZZ-R-proinsuhn during the productron and recovery. (A) SDS-PAGE analysis (reducing conditions) of

ZZ-R-proinsulin at different stages during the recovery process. Lane I. harvested cells. Lane 2, washed sedimented material after

high-pressure homogenization. Lane 3. soluble material after renaturation. Lane 4, ZZ-R-proinsulin after a single-step affinity

purification on IgG-Sepharose. Lane M, marker proteins with molecular masses in kilodaltons indicated at right margin. Coomassie

Brilliant Blue was used for staining. (B) Size exclusion chromatography analysis of IgG-affinity purified ZZ-R-proinsulin. The size

of the major peak corresponds to monomeric ZZ-R-proinsulin and sizes of the minor left peaks correspond to the sizes of dimeric

and multimeric fusion proteins. respectively

1987; Hansson et al., 1994). An expression vector

was constructed encoding a fusion protein com-

prising the two IgG-binding ZZ domains derived

from staphylococcal protein A (Nilsson et al.,

1987) fused to human proinsulin (Fig. 1A). Single basic or dibasic amino acid residues were engi-

neered between the ZZ tail and proinsulin in

addition to the native Arg-Arg and Lys-Arg link-

ers naturally flanking the C-peptide of proinsulin

to allow simultaneous processing of all three sites

by trypsin cleavage (Jonasson et al., 1996). The results in laboratory scale analysis (Jonasson et

al., 1996) suggested that a single arginine-linker

allowed efficient cleavage with trypsin. Thus, the simultaneous recovery of native insulin and C-

peptide and removal of the affinity tag could be

envisioned, and a straightforward production scheme was designed (Fig. 1B).

3.2. Bioreuctor cultivation and recovery qj’ inclusion bodies

An overnight-culture of E. coli cells harbouring

the plasmid pTrpZZ-R-proinsulin was used as

inoculum for a 3 1 bioreactor for production of

ZZ-R-proinsulin. From shake-flask cultivation ex-

periments the fusion protein was found to accu-

mulate intracellularly as inclusion bodies

(Jonasson et al., 1996). After 28 h bioreactor

cultivation (OD 180), with the growth controlled by glycerol feeding, p-indole acrylic acid was

added to the culture in order to induce ZZ-R-

proinsulin production from the trp promoter-reg-

ulated expression system. The culture was

harvested after 3 1 h (OD 210) and the cells were separated from the medium by centrifugation,

giving a dry weight of harvested cells of 46 g/l.


Analysis of harvested cells on SDS-PAGE (Fig. 2, lane 1) showed that ZZ-R-proinsulin (25 kDa) corresponded to - 25% of the observed proteins. The production level of ZZ-R-proinsulin was - 3 g/l culture. This corresponds to an expression level of insulin of - 700 mg/l culture (Table 1). Cell disruption was achieved by high pressure homogenization and the inclusion bodies containing ZZ-R-proinsulin were recovered by centrifugation. The precipitate was washed and SDS-PAGE analysis (Fig. 2A, lane 2) showed that - 90% of the insoluble material corresponded to ZZ-R-proinsulin.

3.3. Solubilization and renaturation

The ZZ-R-proinsulin fusion protein was solubilized and converted to its S-sulfonated form (Cole, 1967; Frank et al., 1981; Cousens et al., 1987) by oxidative sulfitolysis in 8 M urea. Since the ZZ fusion partner does not contain any cysteine residues only the proinsulin will be sulfonated. Non-solubilized material was removed by centrifugation and the supernatant was desalted by dialysis before the renaturation step. Renaturation of the ZZ-R-proinsulin hexasulfonate was then carried out at an approximate protein concentration of 0.8 mg/ml in the presence of a slight excess of /?-mercaptoethanol (Frank et al., 1981). After renaturation, the purity of ZZ-R-proinsulin was analyzed by SDS-PAGE (Fig. 2A, lane 3) which revealed that a considerable purification had been achieved during the solubilization and renaturation steps.

Table 1 Results from the production and recovery process for human insulin and C-peptide

Process step Insulin yield C-peptide yield

(mgil) WY (mg/l) (%)

Cultivation -700 -360 IgG affinity purification 630 90 330 90 Enzymatic clevage 280 44 330 100 Preparative RP-HPLC 150 54 145 44

“Calculated for each process step.

3.4. IgG-affinity chromatography

The ZZ-R-proinsulin was further purified and concentrated by affinity chromatography on IgG-Sepharose. The amount of recovered ZZ- R-proinsulin, 2.7 g/l culture, was estimated by absorbance (A,,, ,.,3 measurements (Table 1) and corresponded well to the SDS-PAGE analysis (Fig. 2A, lane 4) which demonstrated that ZZ-R-proinsulin was stable to proteolysis, pure and produced at high amounts. To determine the percentage of multimeric forms of ZZ-R- proinsulin, a sample of the IgG-affinity purified material was analyzed by size exclusion chromatography (Fig. 2B). Integration of the peak areas on the chromatogram indicated that - 70% of ZZ-R-proinsulin was recovered in the monomeric form. The sizes of the residual material indicated that it contained a mixture of dimeric and multimeric forms of ZZ-R-proinsulin (Fig. 2B).

3.5. Single-step processing of ZZ-R-proinsulin and characterization of the cleavage products

The IgG-affinity purified ZZ-R-proinsulin material was further concentrated by ultrafiltration to a protein concentration of 12 mg/ml. Site-specific cleavage and conversion of proinsulin to native insulin and C-peptide was then performed in a single step by treatment with trypsin and carboxypeptidase B (Kemmler et al., 1971). After completed digestion (30 min), the cleavage products were analyzed by RPC (Fig. 3A, upper). An insulin standard co-migrated with peak III and a C-peptide standard with peak II (data not shown). A peak corresponding to the ZZ fusion partner was identified by incubating the cleavage products with IgG-Sepharose (Fig. 3A, lower). Comparison with the first chromatogram (Fig. 3A, upper), showed that peak I corresponded to the ZZ fusion partner. Analytical-scale analysis of fractions with elution profiles corresponding to peak II and III, respectively, by N-terminal se- quencing and mass spectrometry, has been previously performed (Jonasson et al., 1996), and the experimental results corresponded well to the pre- dictions, demonstrating that the analyzed frac-

248 J. Nilsson et ul. i Journd oJ’ Biotechnology 48 (1996) 241-250

0.6

h 100 15.0 200 25.0

Time (min)

10.0 15.0 20.0

Tune (min)

Fig. 3. Characterization of the products after (A) enzymatic cleavage of affinity purified ZZ-R-proinsulin and (B and C) final

recovery by preparative RP-HPLC. (A) RPC analysis of the ZZ-R-proinsulin cleavage mixtures (upper curve) digested with trypsin

and carboxypeptidase B for 30 min. The bottom curve shows the analysis of the same cleavage mixture after incubation with

IgG-Sepharose. (B and C) RPC chromatography analysis of the human insulin fraction (B) and C-peptide fraction (C), respectively,

recovered by preparative RP-HPLC.

tions indeed contained correctly processed human insulin and C-peptide, respectively.

3.6. Final recovery of’human insulin and

C-peptide

Preparative RP-HPLC on the cleavage solution was performed (data not shown). The fractions containing human insulin and C-peptide were determined by using insulin and C-peptide standards, as described above for the analytical RPC on the cleavage solution. Relevant fractions from the preparative RP-HPLC were collected and subjected to analytical RPC (Fig. 3B and C), demonstrating that both human insulin (Fig. 3B) and its C-peptide (Fig. 3C) were recovered in a pure form. The yield from the preparative RP-HPLC was - 54% for insulin, resulting in a final amount of 300 mg correctly folded material from this 2 1 cultivation. The yield of C-peptide was - 44% resulting in a total of 290 mg C-peptide.

4. Discussion

We have in this study designed and evaluated

a novel process for the production of human insulin and its C-peptide. The rationale was to

produce proinsulin as a fusion protein that was

possible to express at high levels in E. coli, and

in addition to engineer the fusion protein in

such a way that the fusion partner could be

cleaved off simultaneous to the processing of

proinsulin to insulin and C-peptide. Proinsulin

was produced as a fusion to ZZ, derived from

staphylococcal protein A (Nilsson et al., 1987).

This fusion tag was selected due to its stability

to proteolysis, its IgG-binding capacity, its doc- umented high expression levels and solubilizing

properties (Moks et al., 1987; Hansson et al.,

1994; Samuelsson et al., 1991, 1994; UhlCn et al., 1992; Nygren et al., 1994). The chosen pro-

duction strategy allowed the use of an affinity tag for efficient purification (after solubilization

of inclusion bodies and subsequent renatura-

J. Nilsson et al. 1 Journal of Biotechnology 48 (1996) 241-250 249

tion), without the inclusion of additional unit nical Development (NUTEK). We thank Drs. operations for cleavage and removal of the ZZ Maria Murby and Anders Hedrum for help with affinity tag. The tag was demonstrated to be vector constructions, Dr. Henrik Wadensten simultaneously cleaved off with the trypsin/car- (Pharmacia Biopharmaceuticals, Stockholm) for boxypeptidase B digestion of proinsulin to insulin assistance with the preparative RP-HPLC and Dr. and C-peptide, thus resulting in an integrated Luciano Vilela for help. Dr. Tomas Moks is production scheme with rather few unit opera- gratefully acknowledged for fruitful discussions tions (Fig. 1B). and advice.

The yields for each step of the production and recovery process is presented in Table 1. As an initial purification step, IgG affinity chromatography gave high yields (90%) of proteolytically stable and electrophoretically pure ZZ-R-proinsulin (Fig. 2A, lane 4). The single-step trypsin/carboxypeptidase B digestion resulted in very close to 100% yield for the C-peptide, but showed only 44% yield in generating human insulin. The differ- ences in yields are interesting. Since only 70% of the affinity purified proinsulin is monomeric (Fig. 2B), the high yield of native C-peptide suggests that also dimeric and multimeric proinsulin result in C-peptide after trypsin cleavage. In contrast, the data indicate that only the monomeric fraction of proinsulin yields insulin after cleavage. The yields for the final recovery of human insulin and C-peptide by preparative RP-HPLC were 54% and 44%, respectively. Taken together, this indicates that 300 mg correctly folded human insulin and 290 mg C-peptide, respectively, can be recovered from a 2 1 bioreactor cultivation by a recovery scheme involving a very limited number of unit operations.

References

Blight, M.A., Chervaux, C. and Holland, B.I. (1994) Protein secretion pathways in Escherichia coli. Curr. Opin. Bio- technol. 5, 468-474.

Carter, P. (1990) From molecular mechanism to large-scale processes. In: Ladish, M.R., Willson, R.C., Painton, C.- D.C. and Builder, S.E. (Eds.), Protein Purification. Ameri- can Chemical Society Symposium Series, pp. 181- 193.

Chan, S.J., Weiss, J., Konrad, M., White, T., Bahl, C., Yu, S.-D., Marks, D. and Steiner, D.F. (1981) Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli. Proc. Natl. Acad. Sci. USA 78, 540-5405.

Cole, R.D. (1967) Sulfitolysis. Methods Enzymol. 11, 206- 208.

Cousens, L.S., Shuster, J.R., Gallegos, C., Ku, L., Stempien, M.M., Uredea, M.S., Sanches-Pescador, R., Taylor, R. and Tekamp-Olson, P. (1987) High level expression of proinsulin in yeast, Saccharomyces cereoisiae. Gene 61, 2655275.

In conclusion, an integrated process for intracellular production in E. coli of human insulin and its C-peptide is described. The results demon- strate the advantage of using careful genetic design for the production of recombinant biopharmaceuticals. Such strategies will most probably be of increased importance for products with high requirements for cost-effective manufac- turing.

Dalboge, H., Dahl, H.-H.M., Pedersen, J., Hansen, J.W. and Christensen, T. (1987) A novel enzymatic method for production of authentic hGH from an Escherichia coli produced hGH-precursor. Bio/Technology 5, 161- 164.

Datar, R.V., Cartwright, T. and Rosen, C.-G. (1993) Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator. Bio/ Technology 11, 349-357.

deGeus, P., van den Bergh, C.J., Kuipers, O., Verheij, H.M., Hoekstra, W.P.M. and de Haas, G.H. (1987) Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli. Nucl. Acids Res. 15, 3743-3759.

For&erg, G., Baa&up, B., Rondahl, H., Holmgren, E., Pohl, G., Hartmanis, M. and Lake, M. (1992) An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on Des(l-3)insulin-like growth factor I. J. Protein Chem. 11, 201-211.

Acknowledgements

This work was financed by support from the Protein Engineering Program funded by the Swedish National Board for Industrial and Tech-

Frank, B.H., Petee, J.M., Zimmerman, R.E. and Burck, P.J. (1981) The production of human proinsulin and its trans- formation to human insulin and C-peptide. In: Rich, D.H. and Gross, E. (Eds.), Peptides: Synthesis, Structure and Function, Proceedings of the Seventh American Peptide Symposium, Pierce Chemical Company, Rockford, IL, pp. 729-738.

250 J. Nilsson et al. / Journul oj Biotechnology 48 (1996) 241.-250

Guo, L.-H., Stepien, P.P., Tso, J.Y., Brousseau, R., Narang.

S., Thomas, D.Y. and Wu, R. (1984) Synthesis of human

insulin gene. Construction of expression vectors for fused

proinsulin production in Escherichiu coli. Gene 29, 25l

254.

Hansson, M., Stahl, S., Hjorth, R., Uhlen, M. and Moks, T.

(1994) Single-step recovery of a secreted recombinant

protein by expanded bed adsorption. Bio/Technology 12,

2855288.

Jansson, M., Li, Y.-C., Jendeberg, L., Anderson, S., Monte-

hone, G.T. and Nilsson, B. (1996) High level production

of uniformly “N- and ‘C-enriched fusion proteins in

Escherichia coli. J. Biomol. NMR 7, 131~141.

Johansson, B.-L., Sjoberg, S. and Wahren. J. (1992) The

influence of human C-peptide on renal function and glu-

cose utilization in type I (insulin-dependent) diabetic pa-

tients. Diabetologia 35, 12 I 128.

Johansson, B.-L., Kernel], A., Sjdberg, S. and Wahren, J.

(1993) Influence of combined C-peptide and insulin ad-

ministration on renal function and metabolic control in

diabetes type 1. J. Clin. Endocrinol. Metab. 77, 976698 I. Johnson, I. (1983) Human insulin from recombinant DNA

technology. Nature 219, 632-637.

Jonasson, P., Nilsson, J., Samuelsson, E., Moks. T., Stahl.

S. and Uhlin. M. (1996) Single-step trypsin cleavage of a

fusion protein to obtain human insulin and its C-peptide.

Eur. J. Biochem. 236. 6566661

Josephson, S. and Bishop, R. (1988) Secretion of peptides

from E. coli: a production system for the pharmaceutical

industry. Trends Biotechnol. 6, 218-224.

Kang, Y. and Yoon, J.-W. (1994) Effect of modification of

connecting peptide of proinsulin on its export. J. Bio-

technol. 36, 45 -54.

Kemmler, W., Peterson, J.D. and Steiner, D.F. (1971) Stud-

ies on the conversion of proinsulin to insulin. J. Biol.

Chem. 246, 61866791.

Kohler, K., Ljungquist, C., Kondo, A., Veide, A. and

Nilsson, B. (1991) Engineering proteins to enhance their

partitioning coefficients in aqueous two-phase systems.

Bio/Technology 9, 6422646.

Laemmli, U.K. (1970) Cleavage of structural proteins during

the assembly of the head of bacteriophage T4. Nature

227, 680-685.

Moks, T., Abrahmsen, L., Osterlof, B., Josephson. S.,

Ostling, M., Enfors, S.-O., Persson, I., Nilsson, B. and

Uhltn, M. (1987) Large-scale affinity purification of hu-

man insulin-like growth factor I from culture medium of

Escherichia coli. Bio/Technology 5, 3799382.

Murby, M., Cedergren, L., Nilsson, J., Nygren, P.-A., Ham-

marberg, B., Nilsson, B., Enfors, S.-O. and Uhlen, M.

(1991) Stabilization of recombinant proteins from prote-

olytic degradation in Escherichia coli using a dual affinity

fusion strategy. Biotechnol. Appl. Biochem. 14, 336-346.

Murby, M., Nguyen, T.N., Binz, H., UhlCn, M. and Stahl.

S. (1994) Production and recovery of recombinant

proteins of low solubility. In: Pyle, D. (Ed.), Separations

for Biotechnology 3, Bookcraft Ltd., Bath, pp. 336-344.

Nilsson, B., Moks, T.. Jansson, B., Abrahmsen, L., Elmblad,

A.. Holmgren, E., Henrichson, C., Jones, T.A. and Uh-

len, M. (1987) A synthetic IgG-binding domain based on

staphylococcal protein A. Protein Eng. 1, 107- 113.

Nygren, P.-A., Stahl, S. and Uhltn, M. (1994) Engineering

proteins to facilitate bioprocessing. Trends Biotechnol.

12. 184-188.

Olsson, M. and Isaksson, L. (1979) Analysis of rpsD muta-

tions in Escherichiu coli. Mol. Gen. Genet. 169, 251-257.

Rudolph, R. (1995) Successful protein folding on an indus-

trial scale. In: Cleland, J.L. and Craik, C.S. (Eds.), Prin-

ciples and Practice of Protein Folding. John Wiley and

Sons Inc., New York, pp. 2833298.

Samuelsson, E., Wadensten, H., Hartmanis, M., Moks, T.

and Uhlen, M. (1991) Facilitated in vitro refolding of

human recombinant insulin-like growth factor I using a

solubilizing fusion partner. Bio/Technology 9, 363-366.

Samuelsson, E., Moks, T., Nilsson, B. and Uhlen, M. (1994)

Enhanced in vitro refolding of insulin-like growth factor

1 using a solubilizing fusion partner. Biochemistry 33.

4207-4211,

Shen, S.-H. (1984) Multiple joined genes prevent product

degradation in Escherichiu cob. Proc. Nat]. Acad. Sci.

USA 81, 4627-4631.

Stahl. S.J. and Christiansen, L. (1988) Selection for signal

sequence mutations that enhance production of secreted

human proinsulin by Escherichia coli. Gene 71, 147- 156.

Talmadge, K. and Gilbert, W. (1982) Cellular location af-

fects protein stability in Escherichia coli. Proc. Natl.

Acad. Sci. USA 79, 1830- 1833.

Uhlen. M., Forsberg, G., Moks, T., Hartmanis, M. and

Nilsson, B. (1992) Fusion proteins in biotechnology.

Curr. Opin. Biotechnol. 3, 3633369.

Wang, M., Scott, W.A., Rao. R., Udey, J., Conner, G.E.

and Brew, K. (1989) Recombinant bovine lactalbumin

obtained by limited proteolysis of a fusion protein ex-

pressed at high levels in Escherichiu coli. J. Biol. Chem.

264, 21116~21121.

Williams, D.C., Van Frank, R.M., Muth, W.L. and Burnett.

J.P. (1982) Cytoplasmic inclusion bodies in Escherichiu c,oli producing biosynthetic human insulin proteins, Sci-

ence 215, 687-689.

integrated production of human insulin and its c-peptide

Documents