COMMUNICATION TO THE EDITOR

Human Insulin from a PrecursorOverexpressed in the MethylotrophicYeast Pichia pastoris and a SimpleProcedure for Purifying theExpression Product

Yan Wang,1 Zhen-He Liang,1 You-Shang Zhang,1 Shi-Yin Yao,2

Ying-Gao Xu,2 Yue-Hua Tang,1 Shang-Quan Zhu,2 Da-Fu Cui,2

You-Min Feng1,*

1State Key Laboratory of Molecular Biology, Shanghai Institute ofBiochemistry, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences; telephone: 86-21-64374430; fax: 86-21-64338357;e-mail: fengym@sunm.shcnc.ac.cn2Shanghai Institute of Biochemistry, Chinese Academy of Sciences,Shanghai 200031, China

Received 30 May 2000; accepted 15 October 2000

Abstract: The methylotrophic yeast Pichia pastoris,which proved successful in producing many heterolo-gous proteins, was used to express an insulin precursor.A transformant with a high copy number of the geneintegrated into the chromosome was obtained by thedot-blotting method. In high-density fermentation usinga simple culture medium composed mainly of salt andmethanol, the expression level reached 1.5 g/L. A simpletwo-step method was established to purify the expres-sion product from the culture medium with an overallrecovery of about 80%. After tryptic transpeptidation, hu-man insulin with full receptor binding capacity and bio-logical activity was obtained. In the presence of zinc, therecombinant human insulin could be crystallized in therhombohedral form. © 2001 John Wiley & Sons, Inc. Biotech-nol Bioeng 73: 74–79, 2001.Keywords: methylotrophic yeast; insulin precursor; dot-blotting method

INTRODUCTION

Since insulin was discovered about 80 years ago, it has beenstudied extensively in medicine and biochemistry. Nowa-days insulin is still a good model in recent studies of mo-lecular biology, structural biology, signal transduction, andso on. As a drug for treating diabetes, especially IDDM(insulin-dependent diabetes mellitus), insulin is still indis-pensable even though there are several compounds withsome blood sugar-lowering effect.

Human insulin was one of the first proteins expressed in

microorganisms (Ullrich et al., 1977). Since then, recombi-nant human insulin, produced inE. coli (Frank et al., 1981)or yeast (Markussen et al., 1986), has been used instead ofanimal insulin in the clinic. Human insulin was expressed inE. coli as separate chains or proinsulin, but it always formedinclusion bodies that should be dealt with by denaturing andrefolding after fermentation. Yeast was used to minimizesuch handling, in which single-chain monobasic or dibasicprecursors were expressed and the correctly folded productwith correct disulphide bridges was converted to humaninsulin by enzymatic reactions.

[Conventional yeastS. cerevisiaewas usually used toproduce human insulin. However, this system has its ownlimitations, such as difficulty in high-density growth, nopowerful and regulated promoters for expression, etc. So theexpression level was limited. We tried the expression ofinsulin in this yeast, but the level was not high (Zhang et al.,1996). We also triedK. lactis without satisfactory results(Feng et al., 1997).]

The methylotrophic yeastPichia pastorisis a recentlydeveloped system for expressing heterologous proteins. Ithas many advantages, such as the powerful and methanol-regulated AOX1 promoter, the stable expression of the in-tegrated target gene, the high secretory ability, the lowamount of proteins other than the expression product in thecell culture, the easiness of high-density cell growth, and thecheap culture medium needed (Cregg et al., 1993). Thissystem has been used in producing many proteins, with anexpression level ranging from mg/L to g/L. [A preliminaryexpression of insulin in this system and the comparison withthat inS. cerevisiaehave been done, but the potency of the

Correspondence to:Y.-M. FengContract grant sponsor: the Chinese Academy of SciencesContract grant number: KJ951-B1-606.

© 2001 John Wiley & Sons, Inc.

system and purification methods were not fully reported(Kjeldsen et al., 1999).]

Here, we report the high expression of an insulin precur-sor in P. pastorisand a simple procedure for purifying theexpression product. The expression can reach a level of 1.5g/l. A simple two-step procedure for purifying the expres-sion product with high recovery was established. Recombi-nant human insulin obtained from the precursor by tryptictranspeptidation has identical receptor binding capacity andbiological activity as those of native porcine insulin andrecombinant human insulin fromS. cerevisiae.

MATERIALS AND METHODS

Construction of the Plasmid pPIC9K/PIP

The E. coli strain DH5a was used. TheP. pastorisstrainGS115 (his4) and plasmid pPIC9K were from InvitrogenCo. (La Jolla, CA). The plasmid pVT102U/a-MFL-PIPcontaining porcine insulin precursor (PIP) gene anda-mat-ing factor leader (a-MFL) sequence located at the 58 end ofPIP gene was constructed in our laboratory for secretoryexpression of PIP inS. cerevisiae(Zhang et al., 1996). Onlyone residue located at the position of B30 is different fromhuman insulin, Thr, and porcine insulin, Ala. Therefore,porcine insulin can be converted into human insulin bymeans of transpeptidation in the presence of trypsin and Thr(Markussen, 1984). Here, the PIP gene was chemically syn-thesized and the B30 and A1 were connected by a dipeptideAla-Lys. The residue at the position B30 of PIP is Ala, sothe precursor was designated a porcine insulin precursor,also called “insulin precursor.” PIP can be directly con-verted into human insulin by means of transpeptidation(Markussen, 1986; Zhang, 1996). A DNA fragment con-taining the PIP gene and the leader sequence ofa-MFLlocated at the 58 end of PIP was prepared by PCR. The PCRreaction was carried out using plasmid pVT102U/a-MFL-PIP as the template, with the 58 primer ACAGGATCCAT-GAGATTTCCTTCAAT and 38 primer TGAATTCTTC-TAGTTGCAGTAGTTT. The DNA fragment was thencloned into the plasmid pPIC9K between BamH1 andEcoR1 cleavage sites to obtain a plasmid termed pPIC9K/PIP. All DNA manipulations were carried out according tothe standard procedures (Maniatis et al., 1989).

Transformation and Phenotype Identification

The plasmid pPIC9K/PIP was linearized by Bgl II andcloned into theP. pastorisstrain GS115 using the sphero-plast method (Cregg et al., 1985). The transformed cellswere plated onto RDB agar plate without histidine.

Single colonies were grown on the MD plate for furtherhistidine selection and transferred simultaneously onto MDand MM plates. Those that grew fast on the MM plate wereMut+, others were Muts.

Screening Multicopy Transformants Using theDot-Blotting Method

The transformants were screened with the dot-blottingmethod (Clare et al., 1991b). Briefly, the transformantswere cultured in a tube with YPD (yeast extract 1%, poly-pepton 2%, dextrose 2%) in a shaking incubator at 30°C for2 days. Equal aliquots of cells were taken and digested withlyticase (5 U/50ml cell) in 0.5 ml tubes at 37°C for 4 h. Thedigest was adsorbed on a nylon membrane (Hybond-N).After denaturing, the membrane was hybridized with therandom primed32P-labeled PIP (Random Primed LabelingKit, TaKaRa).

Expression of the Insulin Precursor

Small-Scale

The small-scale expression was carried out in a 250 ml flaskor 1 L flask according to the Pichia Expression Kit Manual(Invitrogen) Both Mut+ and Muts transformants were cul-tured. Mut+ was cultured for 4 days and Muts for 7 days.YPD and YPM (yeast extract 1%, polypepton 2%, methanol0.5%) were used in cell culture without and with methanolfor induction, respectively.

Fermentation

High-density fermentation was performed basically accord-ing to Pichia Protocols (Stratton et al., 1998). The followingtwo media were used. Each liter of the basal salt medium(BSM) contained 50 ml glycerol, 26.7 ml H3PO4, 0.93 gCaSO4?2H2O, 18.2 g K2SO4, 14.9 g MgSO4?7H2O, and4.13 g KOH. After sterilization, the pH was adjusted to 5.0with 50% NH4OH. Each liter of the trace element solution(PTM1) contained 6 g CuSO4?5H2O, 0.08 g KI, 3.0 gMnSO4?H2O, 0.2 g (NH4)6Mo7O24?4H2O, 0.02 g H3BO3,0.5 g CaSO4?2H2O, 20 g ZnSO4, 5 ml concentration H2SO4,65 g FeSO4?7H2O, 0.5g CoCl2?6H2O and 0.2 g Biotin.PTM1 solution was sterilized by filtration.

50 ml YPG (yeast extract 1%, polypepton 2%, glycerol2%) in a 250 ml flask was inoculated and cultured in ashaking incubator for 24 h at 30°C as the first seed. Theculture was transferred to 400 ml YPG in two 1-L flasks andcultured for 18–20 h, which was the second seed. The sec-ond seed was added to 5 L BSM and 20 ml PTM1 in a 16-Lfermentor. The pH was adjusted to and maintained at 5.0with 50% NH4OH. After about 20–24 h when glycerol wasdepleted, 50% glycerol containing PTM1 (4 ml/L of 50%glycerol) was fed for 4–6 h, followed by methanol feeding(methanol with 4 ml PTM1 per liter of methanol) last for90–113 h. The culture was harvested when the OD600nm

reached 500 or more. The expression level of the insulinprecursor was 1.5 g/L as determined by HPLC with C8column.

COMMUNICATION TO THE EDITOR 75

Purification of Expression Product by a SimpleTwo-Step Method

After centrifugation (6,000 rpm for 10 min) of the culturedmedium to remove cells, the supernatant was applied on aXAD-7 column and washed with 5% acetic acid and 15%ethanol, 5% acetic acid successively to remove impurities.The expression product was eluted from the column with45% ethanol, 5% acetic acid. The eluent was collected andethanol was removed by rotating evaporation.

The eluent was then loaded on a Sephadex G-50 columnequilibrated with 1 N acetic acid. The insulin precursor waseluted with 1 N acetic acid and freeze-dried.

Conversion of the Precursor into Human Insulin

The transpeptidation reaction was carried out in the pres-ence of Thr(But)-OBut and trypsin as described (Zhang etal., 1996). The reaction mixture was loaded on a SephadexG-50 column to remove the enzyme. The reaction product[B30Thr(But)-OBut] human insulin was purified with aDEAE-Sepharose CL-6B column. The protective group wasdeblocked by trifluoroacetic acid (TFA) to obtain recombi-nant human insulin.

Bioassays of Recombinant Human Insulin

The receptor binding capacity was determined using humanplacental membrane (Feng et al., 1982).

The in vivo biological activity was measured by themouse glucose-lowering method according to the ChinesePharmacopoeia (1995 version).

RESULTS

Cloning and Transformation

The plasmid pPIC9K/PIP was confirmed by DNA sequenc-ing.

Bgl II was used to linearize the plasmid pPIC9K/PIP fortransformation. Since it cleaves the plasmid at two sites, itcan either transplace AOX1 gene in the chromosome toform Muts phenotype or integrate with single crossover toform Mut+ phenotype (Clare et al., 1991a).

Screening High-Copy Number Transformants andIts Fermentation

Using the spheroplast method for transformation, a high-copy strain named P39 was selected. Its copy number wasestimated to be 6–8 (Fig. 1), determined by scanning thedot-blot film and detecting the radioactivity of the cuttingdots, using the mean of the light dots as the value of a singlecopy.

P39 was fermented in a 16-L fermentor using 5 L ofstarting culture medium. The expression level of the insulinprecursor reached 1.5 g/L [(Fig. 2C).]

The time course of expression after methanol feeding isshown in Figure 2.

Two-Step Procedure for Purifying theInsulin Precursor

Hydrophobic chromatography on XAD-7 column is effi-cient to remove pigment and other hydrophilic materialsfrom the broth. The purity of the insulin precursor after gelfiltration through the G-50 column is over 60%. The par-tially purified product can be used in transpeptidation reac-tion to obtain human insulin. Recovery of the two-step pu-rification is more than 80%. After further purification, theproduct is a single band in native PAGE (Fig. 3) and themolecular mass (5958) is consistent with the theoreticalvalue.

Human Insulin from the Precursor andIts Characterization

The conversion of the precursor into human insulin wascarried out by transpeptidation. The HPLC profile of trans-peptidation reaction mixture (Fig. 4) indicates that about80% of the precursor had been converted into[B30Thr(But)-OBut] human insulin. After purification ofthe reaction mixture on Sephadex G-50 and DEAE-Sepharose CL-6B columns, the product [B30Thr(But)-OBut] human insulin was obtained. The protective groupwas deblocked by TFA and removed with ether. The finalproduct of human insulin obtained shows a single band innative PAGE (Fig. 3). It can be crystallized (Fig. 5) in thepresence of zinc.

Bioassay of Human Insulin

The receptor binding capacity, which is identical to nativeporcine or human insulin, is shown in Figure 6. The in vivobiological activity was determined as 27 IU/mg, similar tothat of the recombinant human insulin fromS. cerevisiae.

Figure 1. The graph of dot-blotting for screening of multicopy strains.(→ is P39, the last one is positive control of diluted probe, and the one leftto it is negative control of pPIC9K transformant.)

76 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 73, NO. 1, APRIL 5, 2001

Discussion

Human insulin is one of the earliest recombinant proteins(Ullrich et al., 1977) and the first recombinant protein drugavailable commercially (Bienz-Tademor, 1993). Since insu-

lin is very important not only in medicine but also in basicresearch, it has been expressed in different systems such asE. coli (Frank et al., 1981), yeast (Markussen, 1986; Zhanget al., 1996; Feng et al., 1997; Wang et al., 1998, 1999;Kjeldsen et al., 1999), etc.

Figure 2. High-density fermentation of the strain P39.A: The expression curve rises with cell density after induction. The expression values wereaccording to HPLC with C8 analytical column.B: The expression shown by native PAGE. Each sample with 3m1 ferment supernatant. From left to right1–5: 0 h, 16 h, 40 h, 60 h, 87 h after induction,→ shows the band of PIP.C: The quantification of the ferment supernatant by HPLC. Buffer A: 10% ACN,0.1%TFA; Buffer B: 80% ACN, 0.1%TFA. The samples were eluted with a linear gradient of buffer B (0–55%) in 40 min at a flow rate of 1 ml/min. (a)The standard of the purified insulin precursor 13.5mg/100ml assayed by Folin-phenol method. (b) The final ferment supernatant diluted 9 times with buffer A.

COMMUNICATION TO THE EDITOR 77

P. pastorisis one of the nonconventional yeasts whichhas many advantages for protein expression and recentlyhas been developed as a useful expression system (Cregg etal., 1993). There are many factors affecting the expression

level in P. pastoris(Sreekrishna et al., 1997). Many reportshave shown that high levels of expression are achieved us-ing high copy number strains, although tick anticoagulantpeptide (TAP) (Laroche et al., 1994) and some larger pro-teins reached the level of g/L using a single copy strain. Itis a complicated system and the expression level changeswith different proteins.

We mainly increased the gene dosage to improve expres-sion. For this purpose, a strain P39 with high copy numberwas obtained using Bgl II to linearize the pPIC9K/PIP andcloned into GS115 using the spheroplast method. In ourcase, Bgl II is more efficient to generate high-copy trans-formants than other enzymes such as Sac I or Sal I (Wanget al., 1998). [Small-scale culturing was done to comparethe expression level after screening and the expression in-creased with the gene dosage.]

Another factor for high-level expression is high-densityfermentation. Here, we grew the high copy strain (P39) in a16-L fermentor to express the insulin precursor. The celldensity in terms of OD600nmwas over 500 [when optimizingthe conditions to maintain dissolved O2 above 20%.]

Figure 3. Native PAGE of purified PIP and the final product of humaninsulin. 1. PIP fromS. cerevisiaeas standard. 2. Human insulin fromS.cerevisiaeas standard. 3. PIP fromP. pastoris.4. Human insulin fromP.pastoris.

Figure 4. The HPLC profile of transpeptidation reaction mixture.(*shows the peak of the product [B30Thr(But)-OBut]human insulin.)

Figure 6. Receptor binding activity of human insulin fromP. pastoriscompared with standard human insulin on human placental membrane. (s:human insulin fromP. pastoris;d: standard human insulin.)

Figure 5. The crystals of human insulin fromP. pastoris.

78 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 73, NO. 1, APRIL 5, 2001

Kjeldsen et al. (1999) reported the expression of insulinprecursor in this system using different spacers located be-tween a-MFL and the target gene, as well as a differentleader sequence with the glycosylation sites changed. How-ever, the expression level was not described.

We have established a two-step procedure for purifyingthe expression product according to the composition of theculture and the trait of the product after trying several othermethods, which is simple and with high yield.

References

Bienz-Tademor B. 1993. Biopharmaceuticals go to market: patterns ofworldwide development. Bio/Technol 11:168–172.

Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA. 1991a.High-level expression of tetanus toxin fragment C inPichia pastorisstrains containing multiple tandem integrations of the gene. Bio/Technol 9:455–460.

Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, PayneMM, Sreekrishna K, Henwood CA. 1991b. Production of mouse epi-dermal growth factor in yeast: high-level secretion usingPichia pas-toris strains containing multiple gene copies. Gene 105:205–212.

Cregg JM, Barringer KJ, Hessler AY. 1985.Pichia pastorisas a hostsystem for transformation. Mol Cell Biol 5:3376–3385.

Cregg JM, Vedvick TS, Raschke WC. 1993. Recent advances in the ex-pression of foreign genes inPichia pastoris.Bio/Technol 11:905–910.

Feng YM, Zhu JH, Zhang XT, Zhang YS. 1982. Studies on the mechanismof insulin action VIII. Acta Biochim Biophys Sin 14:137–137.

Feng YM, Zhang BY, Zhang YS, Fukuhara H. 1997. Secretory expressionof porcine insulin precursor inKluyveromyces lactisand its conversioninto human insulin. Acta Biochim Biophys Sin 29:129–134.

Frank BH, Pettee JM, Zimmermann RE, Burck PJ. 1981. The productionof human proinsulin and its transformation to human insulin and C-peptide. In: Rich DH, Gross E, editors. Peptides: synthesis-structure-function. Proceedings of the Seventh American Peptide Symposium,Pierce Chemical Co., Rockford, IL. p 729–738.

Kjeldsen T, Pettersson AF, Hach M. 1999. Secretory expression and char-acterization of insulin inPichia pastoris.Biotechnol Appl Biochem29:79–86.

Laroche Y, Storme V, Meutter JD, Messens J, Lauwereys M. 1994. High-level secretion and very efficient isotopic labeling of tick anticoagulantpeptide (TAP) expression in the methylotrophic yeast,Pichia pastoris.Bio/Technol 12:1119–1124.

Maniatis T, Fritsch EF, Sambrook J. 1989. Molecular cloning: a laboratorymanual (2nd ed.). New York: Cold Spring Harbor Laboratory. 6 p.

Markussen J. 1984. Semisynthesis of human insulin. In: Methods in dia-betes Research (Vol I, Part A). New York: John Wiley & Sons. p404–410.

Markussen J, Damgaard U, Diers I, Fiil N, Hansen MT, Lassen P, NorrisF, Norris K, Schou O, Snel L, Thim L, Voigt HO. 1986. Biosynthesisof human insulin in yeast via single chain precursors. Diabetologia29:568A–569A.

Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, SmithPL, Wierschke JD, Subramaniam A, Birkenberger LA. 1997. Strate-gies for optimal synthesis and secretion of heterologous proteins in themethylotrophic yeastPichia pastoris.Gene 190:55–62.

Stratton J, Chiruvolu V, Meagher M. 1998. High cell-density fermentation.In: Higgins DR, Cregg JM, editors. Pichia protocols (methods in mo-lecular biology, Vol. 103). Totowa. NJ: Human Press. p 107–120.

Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer E, Rutter WJ, GoodmanHM. 1977. Rat insulin genes: construction of plasmids containing thecoding sequences. Science 196:1313–1319.

Wang Y, Liang ZH, Feng YM, Zhang YS. 1998. Secretory expression ofporcine insulin precursor in the methylotrophic YeastPichia pastoris.In: Peptides, biology and chemistry, program & abstracts, InternationalChinese Peptide Symposium, 1998, Lanzhou, China. p 28.

Wang Y, Liang ZH, Zhang YS, Cui DF, Feng YM. 1999. Secretory ex-pression of human insulin in methylotrophic yeastPichia pastoris.Acta Biochim Biophys Sin 31:587–589.

Zhang YS, Hu HM, Cai RR, Feng YM, Zhu SQ, He QB, Tang YH, XuMH, Xu YG, Zhang XT, Liu B, Liang ZH. 1996. Secretory expressionof a single-chain precursor in yeast and its conversion into humaninsulin. Sci China (Series C) 39:225–233.

COMMUNICATION TO THE EDITOR 79