research article expression and purification of c-peptide

Research ArticleExpression and Purification of C-Peptide Containing InsulinUsing Pichia pastoris Expression System

Mohammed N Baeshen12 Thamer A F Bouback1 Mubarak A Alzubaidi1 Roop S Bora13

Mohammed A T Alotaibi1 Omar T O Alabbas1 Sultan M Alshahrani1

Ahmed A M Aljohani1 Rayan A A Munshi1 Ahmed Al-Hejin1 Mohamed M M Ahmed14

Elrashdy M Redwan15 Hassan A I Ramadan16 Kulvinder S Saini13

and Nabih A Baeshen1

1Department of Biological Sciences Faculty of Science King Abdulaziz University PO Box 80203 Jeddah 21589 Saudi Arabia2Department of Biological Sciences Faculty of Science University of Jeddah PO Box 80327 Jeddah 21589 Saudi Arabia3Department of Biotechnology Eternal University Baru Sahib Himachal Pradesh 173 101 India4Nucleic Acids Research Department Genetic Engineering and Biotechnology Research Institute (GEBRI)City of Scientific Research and Technology Applications Alexandria 21934 Egypt5Protein Research Department Genetic Engineering and Biotechnology Research Institute City of Scientific Research andTechnology Applications Alexandria 21934 Egypt6Cell Biology Department Genetic Engineering and Biotechnology Division National Research Centre Tahrir StreetDokki Cairo 12311 Egypt

Correspondence should be addressed to Mohamed M M Ahmed mmmahmed6yahooca

Received 4 May 2016 Accepted 23 June 2016

Academic Editor Maurizio Battaglia Parodi

Copyright copy 2016 Mohammed N Baeshen et alThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countrieshas created a large global market for insulin Moreover exploration of newmethods for insulin delivery including oral or inhalationroute which require very high doses would further increase the demand of cost-effective recombinant insulin Various bacterialand yeast strains have been optimized to overproduce important biopharmaceuticals One of the approaches we have taken is theproduction of recombinant human insulin along with C-peptide in yeast Pichia pastorisWe procured a cDNA clone of insulin fromOrigene Inc USA Insulin cDNAwas PCR amplified and cloned into yeast vector pPICZ-120572 Cloned insulin cDNAwas confirmed byrestriction analysis and DNA sequencing pPICZ-120572-insulin clone was transformed into Pichia pastoris SuperMan5 strain SeveralZeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulinspecific primers Expression of insulin in Pichia clones was confirmed by ELISA SDS-PAGE and Western blot analysis In vivoefficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin In conclusion a biologicallyactive human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system

1 Introduction

Diabetes mellitus is a metabolic disorder which is character-ized by hyperglycemia due to low level or complete deficiencyof insulin hormone Type 1 diabetes is triggered by insulindeficiency whereas in most cases type 2 diabetes is causedby insulin resistance along with insufficient insulin secretionA chronic hyperglycemic condition is more often associatedwith vascular complications particularly involving the eyes

kidneys hearts blood vessels and nerves [1] Presentlydiabetes is one of the leading causes of death globally [2]

Insulin is a hormone which is synthesized by the 120573 cellsof the pancreatic islets of Langerhans It plays a key role inmodulating blood glucose levels It is produced as a 108-amino acid long single polypeptide precursor preproinsulincontaining a signal sequence at its N-terminus which iscleaved by a specific endopeptidase Proinsulin is furtherproteolytically processed in the coated secretory granules

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3423685 7 pageshttpdxdoiorg10115520163423685

2 BioMed Research International

producing a mature insulin and 34 amino acids of C-peptideMature insulin has molecular weight of 5807 daltons and iscomprised of two polypeptides connected by two interchaindisulphide bondsTheA chain is composed of 21 amino acidswhereas the B chain contains 30 amino acids [3 4]

The initial approach for commercial production of re-combinant human insulin employed by scientists in Genen-tech required transformation of the cDNA encoding for thehuman insulin A and B chains separately into E coli cells(K12) These cells were then cultured individually in largefermenters and both insulin peptides were purified usingchromatographic methods The A and B chains of insulinwere then incubated together under oxidizing conditions topromote folding and interchain disulphide bond formationhence forming an active human insulin [5 6] An alterna-tive approach adopted by Eli Lilly Research Laboratoriesinvolved expression of a single polypeptide of human proin-sulin in E coli followed by purification and removal of C-peptide by proteolytic cleavage to yield the active insulin[7 8] Currently E coli and Saccharomyces cerevisiae arethe preferred expression host for commercial productionof recombinant human insulin for the therapeutic use inhumans [9ndash11]

The main objective of our work is to explore other alter-nate hosts such as Pichia pastoris for large scale productionof recombinant human insulin Pichia pastoris like othereukaryotes offer several advantages including proper proteinfolding protein processing and posttranslational modifica-tions Moreover like E coli and Saccharomyces cerevisiae itis very easy to manipulate genetically and very cost-effectiveexpression system for bulk production of recombinant pro-teins Pichia pastoris is a methylotrophic yeast which metab-olizes methanol as a carbon source Methanol is oxidized toformaldehyde by the activity of the enzyme alcohol oxidase(AOX1) in presence of oxygen Since alcohol oxidase haspoor affinity for oxygen cells compensate it by producinglarge amount of the enzyme Expression of alcohol oxidaseis induced by methanol to very high levels approximatelymore than 30 of total protein in cytosol of Pichia cellsThe promoter controlling the synthesis of alcohol oxidaseis used for expression of recombinant proteins in Pichiapastoris [12 13] Besides high expression levels Pichia pastorishas another significant advantage in glycosylation pattern ofheterologous protein in comparison to Saccharomyces cere-visiae It has been shown that heterologous proteins in Pichiaare not hyperglycosylated as length of the oligosaccharidechains added to proteins is around 8ndash14 mannose residuesper side chain in Pichia which is very short as comparedto that in S cerevisiae that is approximately sim50 to 150mannose residues per side chain [14 15] Moreover unlikein Pichia glycosylated proteins produced from S cerevisiaehave terminal 12057213 glycan linkages which are responsiblefor the hyperantigenic property of these proteins and thusrendering them unsuitable for therapeutic applications inhuman [16] Hence recombinant glycoprotein produced inPichia may be more similar to the glycoprotein structureof higher eukaryotes These important features make Pichiaan attractive expression host for large scale production ofrecombinant proteins for therapeutic use

2 Materials and Methods

21 Cloning of Insulin cDNA in Pichia Vector PlasmidpCMV6-XL harboring proinsulin cDNA was procured fromOrigene Inc USA and used as a template to amplify insulincDNA For secreted expression of insulin there are forwardprimer 51015840-tgatgaattctttgtgaaccaacacctgtgcgg-31015840 and reverseprimer 51015840-acttctcgagagttgcagtagttctccagctggta-31015840 ProinsulincDNA was PCR amplified using insulin specific primersand High Fidelity pfu PCR amplified proinsulin cDNA wasdouble-digested with EcoRI and XhoI and ligated with thepPICZ-alpha vector double-digested with EcoRI and XhoILigated mixture was transformed into competent E coli cellsSeveral Zeocin resistant transformants were obtained on LB-Zeocin plate Few clones were screened for the presenceof proinsulin cDNA in pPICZ-alpha vector by restrictionmapping Proinsulin cDNA cloned in Pichia vector pPICZ-120572 was further confirmed by DNA sequencing

22 Transformation of Pichia pastoris pPICZ-120572-proinsulinconstruct was transformed into Pichia pastoris SuperMan5strain by electroporation For efficient integration of recombi-nant construct into Pichia genome pPICZ-120572-insulin plasmidDNAwas linearized with SacI Pichia strain was transformedwith the insulin construct by electroporation as instructed bymanufacturer Briefly 05mL of overnight grown culture ofPichia pastoris SuperMan5 strain was inoculated into 500mLof YPDmedia in a 2-liter flask and allowed to grow at 30∘C toan OD

600sim 13ndash15 Cells were centrifuged at 1500timesg at 4∘C

for 5min and cell pellet waswashedwith 250mLof sterile ice-cold water twice and once with 20mL of ice-cold 1M sorbitolCells were centrifuged at 1500timesg at 4∘C for 5min and cellpellet was resuspended in 1mL of ice-cold 1M sorbitol andkept on ice for 10min 100 120583L of cells was mixed with 10 120583gof linearized pPICZ-insulin plasmid DNA and transferred toan ice-cold 02 cm electroporation cuvette and kept on icefor 5min Cells were electroporated at 540V 25120583F resistanceset to infinite (infin) and pulse time of 15ndash20ms Immediately1mL of ice-cold 1M sorbitol was added to the cuvette andcells were transferred to a sterile 15mL tube and incubatedat 30∘C without shaking for 2 hrs 50ndash100120583L cell suspensionwas plated on YPDS plates containing 100120583gmL Zeocinincubated at 30∘C for 3ndash5 days until colonies form Forselecting putative multicopy recombinants transformationmix was plated on increasing concentration of Zeocin thatis 500ndash2000120583gmL

23 Expression and Purification of Insulin Protein Using asingle colony 25mL of MGYH media (minimal mediumcontaining glycerol and histidine) was inoculated in a 250mLbaffled flask and grown at 28ndash30∘C in a shaking incuba-tor (250ndash300 rpm) until culture reaches an OD

600= 2ndash6

(approximately 16ndash18 hours) The cells were centrifuged at1500ndash3000timesg for 5min at room temperature Supernatantwas discarded and cell pellet was resuspended to an OD

600

of 10 in MMH (minimal medium containing methanol)medium to induce expression (approximately 100ndash200mL)Culture was placed in a 1-liter baffled flask and kept inincubator 100methanol was added to a final concentration

BioMed Research International 3

of 05 methanol after every 24 hours for induction At thevarious time interval 1mL of the culture was transferred toa 15mL microcentrifuge tube These samples were used toanalyze expression levels of proinsulin and also determine theoptimal time postinduction to harvest the cells Cells wereharvested in a tabletop microcentrifuge for 2-3 minutes atroom temperature For secreted expression the supernatantwas transferred to a separate tube The supernatant and thecell pellets were stored at minus80∘C until further use Insulinprotein concentration in the culture supernatant before andafter induction was estimated by using micro protocol ofPierce Coomassie (Bradford) Protein Assay Kit accordingto Thermo Scientific instruction The cell pellets and super-natants were analyzed for protein expression by SDS-PAGEWestern blot and ELISA

24 ELISA The in-house expressed insulin and commercialinsulin were used for coating the Costar ELISA plates incarbonatebicarbonate buffer (pH 96) for 1 hr at 37∘C andfollowed by incubation at 4∘C for overnight The plates wereblocked with 1 BSA + 01 tween 20 Proteins were detectedby using specific monoclonal antibody against recombinanthuman insulin (Takara Otsu Shiga Japan) at dilution1 1000 in washing buffer for 1 hr and then reacted withgoat anti-mouse-alkaline phosphatase conjugated antibodyfor 1 hr at dilution 1 2000 in washing buffer 50120583Lwell of p-nitrophenyl phosphate (pNPP) substrate was added to eachwelland reading was taken at 495 nm [11]

25 In Vivo Efficacy Studies in Streptozotocin InducedDiabeticMice 50 seven-week-old Swiss outbred mice having weightof 18ndash20 gramsmice were used in this study Mice weredivided into 5 groups each group comprising of 10 mice(5 females and 5 males in separate cages) as follows (1)Control Group I (no treatment) (2) Control Group II (strep-tozotocin + commercial insulin NovoRapid FlexPen) (3)Control Group III (streptozotocin no insulin treatment) (4)Experimental Group IV (streptozotocin + proinsulin pro-duced in the lab by yeast after 144 hours) (5) ExperimentalGroup V (no streptozotocin proinsulin produced in thelab by yeast after 144 hours) All the animals were kept intheir cages for 5ndash7 days before streptozotocin injection Theglucose level was estimated in all animals before injectionDiabetes was induced by using low dose of streptozotocinin mice (175mgmice) for Groups II III and IV and allthe animals were kept for 10ndash12 days before treatment withthe insulin products The glucose level was estimated in allanimals before treatment with insulin products Antidiabeticagents that is commercial insulin for Group II and humanproinsulin for Groups IV and V were injected into respectiveanimals (injection by 06U100 g) The glucose level wasestimated in all five groups after 2 4 and 6 hrs

3 Results and Discussion

31 Cloning of Insulin cDNA in Pichia Vector pPICZ-120572 Plas-mid pCMV6-XL harboring proinsulin cDNA was procuredfrom Origene Inc USA Insert DNA (approx 500 bp) was

confirmed by restriction digestion with Not I as there aretwo sites for Not I flanking the insert DNA This clone wasused as a template to amplify the proinsulin cDNA A 257 bpproinsulin cDNA was PCR amplified using insulin specificprimers and High Fidelity pfu (Figure 1(b)) PCR amplifiedproinsulin cDNA was cloned in pPICZ-120572 under the controlof strong and inducible AOX1 promoter Moreover proin-sulin gene was cloned in frame with the native S cerevisiae120572-factor signal sequence at N-terminus and polyhistidinetag at C-terminus to facilitate the efficient secretion andpurification of recombinant protein as shown in Figure 1(a)Ligated mixture was transformed into competent E coli cellsSeveral Zeocin resistant transformants were obtained on LB-Zeocin plate Few clones were screened for the presence ofproinsulin cDNA in pPICZ vector Four clones were foundto be positive as shown in Figure 2(a) These +ve cloneswere further confirmed by PCR using insulin specific primers(Figure 2(b)) and DNA sequencing

32 Transformation of Pichia pastoris Pichia pastoris Super-Man5 strain was selected for expression of insulin protein Inthis GlycoSwitch strain glycan processing enzyme (OCH1)has beenmutated to prevent glycan elongation andmoreoverit overexpresses a heterologous mannosidase to cleave extraglycans to generate homogenous Man5 structure This par-ticular strain of Pichia synthesizes recombinant protein withmore human-like glycosylation and hence it is more suitablefor production of therapeutic proteins Linearized form ofpPICZ-120572-insulin plasmid DNA was transformed into Pichiapastoris SuperMan5 strain by electroporation Since Pichiaexpression vectors do not contain yeast origin of replicationlinearization of plasmid facilitates efficient recombinationand integration of DNA in Pichia genome For selectingputativemulticopy recombinants transformantswere furthercultured in presence of very high concentration of Zeocinthat is 1000 and 2000120583gmL Zeocin Integration of insulincDNA in Pichia genome was further confirmed by PCR asshown in Figure 3

33 Expression and Purification of Insulin Protein Expressionof insulin protein in Pichia clones was analyzed by ELISAand Western blot analysis using insulin specific monoclonalantibody As insulin was fused with the yeast 120572ndashmating factorsignal sequence at the N-terminus recombinant insulinwas detected in sufficient amount in culture supernatant(Figure 4) Expression of insulin was detected in Pichiaclones by ELISA as well as by SDS-PAGE analysis as seenin Table 1 and Figures 4 and 5 As the Western blot imagerevealed the expressed insulin had higher molecular weight(sim8KDa) in comparison with the standard one due to thepresence of C-chain in the recombinant insulin The SMUTsclone was observed to be superior over +MUT P pastorisclone for expression The expressed and secreted insulinprotein concentration as estimated with Bradford methodswas 5mgL after 144 hrs of induction (Table 1)

34 Evaluation of the Activity of Recombinant Human Insulinin Diabetic Mice Biological activity of recombinant human


5998400 AOX1

promoter

120572-factorsignal

sequence Proinsulin cDNA 6X HIS AOX1 TT

C-peptide

S S

CN

SSS

S

A chain

B chain

Zeocinresistance

gene

(a)

1 2 3 4 5

257 bpproinsulin

cDNA

(b)

Figure 1 (a) Schematic diagram showing construction of recombinant pPICZ-120572-proinsulin plasmid (b) Agarose gel electrophoresis showingPCR amplification of proinsulin cDNA Lane 1 DNA marker lanes 2ndash5 PCR amplified proinsulin cDNA

1 2 3 4 5 6 7 8 9 10

14 15 16 17 18 19 20 21 22 23

pPICZ-120572 (36 kb)

257 bpproinsulin

cDNA

257 bpproinsulin

cDNA

(a)

1 2 3 4

257 bpproinsulin

cDNA

(b)

Figure 2 (a) Agarose gel electrophoresis for screening of positive E coli clones harboring recombinant pPICZ-120572-proinsulin Lanes 1 and 14DNA marker lanes 2 to 13 and 15 to 26 plasmid DNA isolated from Zeocin resistant E coli transformants and double-digested with EcoRIand XhoI (b) Agarose gel electrophoresis for confirmation of clones by PCR Lane 1 DNA marker lanes 2ndash5 positive clones 1 to 4


1 2 3 4 5 6 7 8 9 10

257 bpproinsulin

cDNA

Figure 3 Agarose gel electrophoresis for checking the integration of proinsulin cDNA into Pichia genome Transformation of pPICZ-C-insulin into Pichia pastoris SuperMan5 strain was done by electroporation and confirmation for integration of insulin cDNA in Pichia genomewas done by PCR Lanes 1 to 10 recombinant Pichia clones harboring pPICZ-120572-proinsulin plasmid

5

10

15

25

35

40

55

70

100

130

170kDa

1 2 3 4 5 6 7 8 9 10 M

ProinsulinCommercial insulin

Figure 4 SDS-PAGE gel showing expression of recombinant human proinsulin in Pichia pastoris Standard insulin (lane 10) and expressedhuman proinsulin at various time intervals (lanes 1ndash9) in Pichia pastoris were run in 15 SDS-PAGE About 10 120583L of transformed P pastorisculture supernatants was loaded into lanes 1ndash9 As the image revealed the expressed proinsulin had higher molecular weight (sim8KDa) incomparison with the standard one (lane 10) as it contains the C-peptideThe sim8KDa band intensity seems increasing with the time intervals24 h to 96 h as shown from lanes 9 to 1

Proinsulin

1 2 3 4 5 6 7 8 9 10

Commercialinsulin

(a)

Proinsulin

1 2 3 4 5 6 7 8 9 10

(b)

Figure 5 Western blot analysis to confirm the secreted expression of proinsulin in Pichia pastoris (a) SDS-PAGE gel showing purifiedproinsulin protein (b) Western blot analysis using insulin specific monoclonal antibodies

insulin produced in Pichia pastoriswas checked in streptozo-tocin induceddiabeticmiceOur data indicated the efficacy ofrecombinant insulin in lowering the glucose level in diabeticmice as shown in Figure 6 (Group IV and Group V animals)Since recombinant proteins expressed in Pichia are properlyfolded human insulin with intact C-peptide was found to befunctionally activeHowever inGroup II glucose levels could

not be monitored as all the animals died in this particulargroup

Recombinant human insulin has been commerciallyproduced using either E coli or Saccharomyces cerevisiaeHowever increasing cases of diabetes all over the worldand development of new mode of insulin delivery suchas oral or inhalation have led to increase in demand for


Table 1 Expression of recombinant human insulin in Pichia pastoris as evaluated by ELISA

Commercial insulinTime (hrs)

Recombinant proinsulinProtein conc(120583gmL) OD 495 Mut+ clone MutS clone

OD 495 Protein conc (120583gmL) OD 495 Protein conc (120583gmL)5 0695 plusmn 0031 0 0447 plusmn 0015 mdash 0473 plusmn 0020 mdash10 0706 plusmn 0015 24 0523 plusmn 0010 8 0587 plusmn 0120 1115 0776 plusmn 0015 72 0749 plusmn 0023 20 00818 plusmn 0021 2620 0798 plusmn 0070 96 1155 plusmn 0130 31 1205 plusmn 0101 3525 0802 plusmn 0110 144 0870 plusmn 0030 23 1015 plusmn 0060 2930 0870 plusmn 009Mut+ methanol utilization +ve cloneMutS methanol utilization slow phenotype

0

20

40

60

80

100

120

140

Glu

cose

conc

(m

gdL

)

Afte

r 7 d

ays o

fin

habi

tatio

n

Afte

r 10

days

of S

TZin

ject

ion

Afte

r 2 h

rs o

f ins

ulin

treat

men

t

Afte

r 4 h

rs o

f ins

ulin

treat

men

t

Afte

r 6 h

rs o

f ins

ulin

treat

men

t

GI not treatedGII STZ+ commercial insulinGIII STZ

GIV STZ+ recombinant insulinGV recombinant insulin

Figure 6 Evaluation of the efficacy of recombinant human insulinin diabetic mice G group STZ streptozotocin

recombinant insulin There is an utmost need to improve theproduction capacity of current manufacturing technologiesand also explore alternate expression host which can providehigher yield of recombinant insulin Transgenic plants havepotential to produce large amount of insulin at very low costBiologically active insulin has been shown to be expressedat very high levels in seeds or leaves of transgenic plantswhich provide additional advantage of long-term storage [1718] In this study we have explored the possibility of usingmethylotropic yeast Pichia pastoris for producing recombi-nant insulin Several studies have reported the tremendouspotential of Pichia pastoris to produce large amount offunctionally active heterologous proteins [19ndash23] Moreoverrecombinant proteins are not hyperglycosylated in Pichiawhich is seen as amajor concern for the recombinant proteinsbeing produced in Saccharomyces cerevisiae Moreover wehave expressed the proinsulin along with the C-peptidewhich is not present in the commercially available insulin C-peptide had been previously thought to be biologically inac-tive but few studies in last decade have shown the significanceof C-peptide in treatment of diabetic complications such as

nerve stimulation and renal functions C-peptide had beenshown to act independently of mature insulin by activatingG-protein which causes influx of intracellular Ca2+ due toopening of Ca2+ channels and thus leads to activation ofendothelial nitric oxide synthase Na+K+-ATPase and MAPkinase pathway [24] Activation of Na+K+-ATPase playsimportant role in restoration of renal function in diabeticpatients [25] and activity of endothelial nitric oxide synthaseand Na+K+-ATPase is very essential for nerve function [26]Boyhan and Daniell had reported the expression of humanproinsulin containing all the three peptides that is A- B-and C-peptides in tobacco leaves with approximate yieldof 3mgg leaf Delivery of proinsulin into mice resulted inreduction in blood glucose levels which was comparable tothe commercial insulin [17] Recent studies have suggestedthat inclusion of C-peptide would be more beneficial in long-term treatment of diabetic complications including impairednerve and renal functions [24 26]

4 Conclusion

In conclusion we report here the expression and purificationof biologically active proinsulin along with C-peptide whichoffer some advantage over currently available insulins inmanaging long-term complications of diabetesMoreover weare in process of encapsulating the recombinant insulin innanoparticles to develop oral insulin

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

All authors contributed in writing designing and perform-ing studies All authors read and approved the final paper

Acknowledgments

This work was supported by the NSTIP strategic technologiesprogram in the Kingdom of Saudi Arabia (Project no10-BIO1257-03) The authors also acknowledge assistance


from the Science amp Technology Unit Deanship of ScientificResearch and Deanship of Graduate Studies King AbdulazizUniversity Jeddah KSA

References

[1] S Z Swidan and P A Montgomery ldquoEffect of blood glucoseconcentrations on the development of chronic complications ofdiabetes mellitusrdquo Pharmacotherapy vol 18 no 5 pp 961ndash9721998

[2] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[3] Z Vajo J Fawcett and W C Duckworth ldquoRecombinantDNA technology in the treatment of diabetes insulin analogsrdquoEndocrine Reviews vol 22 no 5 pp 706ndash717 2001

[4] G Walsh ldquoTherapeutic insulins and their large-scale manufac-turerdquo Applied Microbiology and Biotechnology vol 67 no 2 pp151ndash159 2005

[5] R E Chance and J A Hoffmann ldquoProcess for producing aninsulinrdquo US Patent 4421685 1983

[6] R E Chance and B H Frank ldquoResearch development produc-tion and safety of biosynthetic human insulinrdquo Diabetes Carevol 16 no 3 pp 133ndash142 1993

[7] N A Baeshen M N Baeshen A Sheikh et al ldquoCell factoriesfor insulin productionrdquo Microbial Cell Factories vol 13 no 1article 141 pp 1ndash9 2014

[8] R Chance N Glazer and K Wishner ldquoInsulin lispro (Huma-log)rdquo in Biopharmaceuticals an Industrial Perspective G Walshand B Murphy Eds pp 149ndash172 Kluwer Dordrecht TheNetherlands 1999

[9] M N Baeshen A M Al-Hejin R S Bora et al ldquoProductionof biopharmaceuticals in E Coli current scenario and futureperspectivesrdquo Journal ofMicrobiology andBiotechnology vol 25no 7 pp 953ndash962 2015

[10] E M Redwan M M Ahmed G Abd EL-Aziz and A Aboul-Enein ldquoSynthesis of the human insulin gene assembling andamplificationrdquo Arab Journal of Biotechnology vol 9 pp 265ndash272 2006

[11] E-R M Redwan S M Matar G A El-Aziz and E ASerour ldquoSynthesis of the human insulin gene protein expres-sion scaling up and bioactivityrdquo Preparative Biochemistry andBiotechnology vol 38 no 1 pp 24ndash39 2008

[12] S B Ellis P F Brust P J Koutz A F Waters M M Harpoldand T R Gingeras ldquoIsolation of alcohol oxidase and two othermethanol regulatable genes from the yeast Pichia pastorisrdquoMolecular and Cellular Biology vol 5 no 5 pp 1111ndash1121 1985

[13] J F Tschopp G Sverlow R Kosson W Craig and L GrinnaldquoHigh-level secretion of glycosylated invertase in the methy-lotrophic yeast pichia pastorisrdquo BioTechnology vol 5 no 12pp 1305ndash1308 1987

[14] L S Grinna and J F Tschopp ldquoSize distribution and gen-eral structural features of N-linked oligosaccharides from themethylotrophic yeast Pichia pastorisrdquo Yeast vol 5 no 2 pp107ndash115 1989

[15] J F Tschopp P F Brust J M Cregg C A Stillman and TR Gingeras ldquoExpression of the lacZ gene from two methanol-regulated promoters in Pichia pastorisrdquo Nucleic Acids Researchvol 15 no 9 pp 3859ndash3876 1987

[16] J M Cregg T S Vedvick and W C Raschke ldquoRecentadvances in the expression of foreign genes in Pichia pastorisrdquoBioTechnology vol 11 no 8 pp 905ndash910 1993

[17] D Boyhan and H Daniell ldquoLow-cost production of proinsulinin tobacco and lettuce chloroplasts for injectable or oral deliveryof functional insulin and C-peptiderdquo Plant Biotechnology Jour-nal vol 9 no 5 pp 585ndash598 2011

[18] C L Nykiforuk J G Boothe E W Murray et al ldquoTransgenicexpression and recovery of biologically active recombinanthuman insulin from Arabidopsis thaliana seedsrdquo Plant Biotech-nology Journal vol 4 no 1 pp 77ndash85 2006

[19] C Gurramkonda S Polez N Skoko et al ldquoApplication ofsimple fed-batch technique to high-level secretory productionof insulin precursor using Pichia pastoris with subsequentpurification and conversion to human insulinrdquo Microbial CellFactories vol 9 article 31 2010

[20] T Kjeldsen A F Pettersson and M Hach ldquoSecretory expres-sion and characterization of insulin in Pichia pastorisrdquo Biotech-nology and Applied Biochemistry vol 29 no 1 pp 79ndash86 1999

[21] T Kjeldsen ldquoYeast secretory expression of insulin precursorsrdquoApplied Microbiology and Biotechnology vol 54 no 3 pp 277ndash286 2000

[22] MMansur C Cabello L Hernandez et al ldquoMultiple gene copynumber enhances insulin precursor secretion in the yeastPichiapastorisrdquo Biotechnology Letters vol 27 no 5 pp 339ndash345 2005

[23] Y Wang Z-H Liang Y-S Zhang et al ldquoHuman insulin froma precursor overexpressed in the methylotrophic yeast Pichiapastoris and a simple procedure for purifying the expressionproductrdquo Biotechnology and Bioengineering vol 73 no 1 pp74ndash79 2001

[24] J Wahren ldquoC-peptide new findings and therapeutic implica-tions in diabetesrdquo Clinical Physiology and Functional Imagingvol 24 no 4 pp 180ndash189 2004

[25] L Rebsomen A Khammar D Raccah and M Tsimaratos ldquoC-Peptide effects on renal physiology and diabetesrdquo Experimentaldiabetes research vol 2008 Article ID 281536 2008

[26] J Wahren K Ekberg and H Jornvall ldquoC-peptide is a bioactivepeptiderdquo Diabetologia vol 50 no 3 pp 503ndash509 2007

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producing a mature insulin and 34 amino acids of C-peptideMature insulin has molecular weight of 5807 daltons and iscomprised of two polypeptides connected by two interchaindisulphide bondsTheA chain is composed of 21 amino acidswhereas the B chain contains 30 amino acids [3 4]

The initial approach for commercial production of re-combinant human insulin employed by scientists in Genen-tech required transformation of the cDNA encoding for thehuman insulin A and B chains separately into E coli cells(K12) These cells were then cultured individually in largefermenters and both insulin peptides were purified usingchromatographic methods The A and B chains of insulinwere then incubated together under oxidizing conditions topromote folding and interchain disulphide bond formationhence forming an active human insulin [5 6] An alterna-tive approach adopted by Eli Lilly Research Laboratoriesinvolved expression of a single polypeptide of human proin-sulin in E coli followed by purification and removal of C-peptide by proteolytic cleavage to yield the active insulin[7 8] Currently E coli and Saccharomyces cerevisiae arethe preferred expression host for commercial productionof recombinant human insulin for the therapeutic use inhumans [9ndash11]

The main objective of our work is to explore other alter-nate hosts such as Pichia pastoris for large scale productionof recombinant human insulin Pichia pastoris like othereukaryotes offer several advantages including proper proteinfolding protein processing and posttranslational modifica-tions Moreover like E coli and Saccharomyces cerevisiae itis very easy to manipulate genetically and very cost-effectiveexpression system for bulk production of recombinant pro-teins Pichia pastoris is a methylotrophic yeast which metab-olizes methanol as a carbon source Methanol is oxidized toformaldehyde by the activity of the enzyme alcohol oxidase(AOX1) in presence of oxygen Since alcohol oxidase haspoor affinity for oxygen cells compensate it by producinglarge amount of the enzyme Expression of alcohol oxidaseis induced by methanol to very high levels approximatelymore than 30 of total protein in cytosol of Pichia cellsThe promoter controlling the synthesis of alcohol oxidaseis used for expression of recombinant proteins in Pichiapastoris [12 13] Besides high expression levels Pichia pastorishas another significant advantage in glycosylation pattern ofheterologous protein in comparison to Saccharomyces cere-visiae It has been shown that heterologous proteins in Pichiaare not hyperglycosylated as length of the oligosaccharidechains added to proteins is around 8ndash14 mannose residuesper side chain in Pichia which is very short as comparedto that in S cerevisiae that is approximately sim50 to 150mannose residues per side chain [14 15] Moreover unlikein Pichia glycosylated proteins produced from S cerevisiaehave terminal 12057213 glycan linkages which are responsiblefor the hyperantigenic property of these proteins and thusrendering them unsuitable for therapeutic applications inhuman [16] Hence recombinant glycoprotein produced inPichia may be more similar to the glycoprotein structureof higher eukaryotes These important features make Pichiaan attractive expression host for large scale production ofrecombinant proteins for therapeutic use

2 Materials and Methods

21 Cloning of Insulin cDNA in Pichia Vector PlasmidpCMV6-XL harboring proinsulin cDNA was procured fromOrigene Inc USA and used as a template to amplify insulincDNA For secreted expression of insulin there are forwardprimer 51015840-tgatgaattctttgtgaaccaacacctgtgcgg-31015840 and reverseprimer 51015840-acttctcgagagttgcagtagttctccagctggta-31015840 ProinsulincDNA was PCR amplified using insulin specific primersand High Fidelity pfu PCR amplified proinsulin cDNA wasdouble-digested with EcoRI and XhoI and ligated with thepPICZ-alpha vector double-digested with EcoRI and XhoILigated mixture was transformed into competent E coli cellsSeveral Zeocin resistant transformants were obtained on LB-Zeocin plate Few clones were screened for the presenceof proinsulin cDNA in pPICZ-alpha vector by restrictionmapping Proinsulin cDNA cloned in Pichia vector pPICZ-120572 was further confirmed by DNA sequencing

22 Transformation of Pichia pastoris pPICZ-120572-proinsulinconstruct was transformed into Pichia pastoris SuperMan5strain by electroporation For efficient integration of recombi-nant construct into Pichia genome pPICZ-120572-insulin plasmidDNAwas linearized with SacI Pichia strain was transformedwith the insulin construct by electroporation as instructed bymanufacturer Briefly 05mL of overnight grown culture ofPichia pastoris SuperMan5 strain was inoculated into 500mLof YPDmedia in a 2-liter flask and allowed to grow at 30∘C toan OD

600sim 13ndash15 Cells were centrifuged at 1500timesg at 4∘C

for 5min and cell pellet waswashedwith 250mLof sterile ice-cold water twice and once with 20mL of ice-cold 1M sorbitolCells were centrifuged at 1500timesg at 4∘C for 5min and cellpellet was resuspended in 1mL of ice-cold 1M sorbitol andkept on ice for 10min 100 120583L of cells was mixed with 10 120583gof linearized pPICZ-insulin plasmid DNA and transferred toan ice-cold 02 cm electroporation cuvette and kept on icefor 5min Cells were electroporated at 540V 25120583F resistanceset to infinite (infin) and pulse time of 15ndash20ms Immediately1mL of ice-cold 1M sorbitol was added to the cuvette andcells were transferred to a sterile 15mL tube and incubatedat 30∘C without shaking for 2 hrs 50ndash100120583L cell suspensionwas plated on YPDS plates containing 100120583gmL Zeocinincubated at 30∘C for 3ndash5 days until colonies form Forselecting putative multicopy recombinants transformationmix was plated on increasing concentration of Zeocin thatis 500ndash2000120583gmL

23 Expression and Purification of Insulin Protein Using asingle colony 25mL of MGYH media (minimal mediumcontaining glycerol and histidine) was inoculated in a 250mLbaffled flask and grown at 28ndash30∘C in a shaking incuba-tor (250ndash300 rpm) until culture reaches an OD

600= 2ndash6

(approximately 16ndash18 hours) The cells were centrifuged at1500ndash3000timesg for 5min at room temperature Supernatantwas discarded and cell pellet was resuspended to an OD

600

of 10 in MMH (minimal medium containing methanol)medium to induce expression (approximately 100ndash200mL)Culture was placed in a 1-liter baffled flask and kept inincubator 100methanol was added to a final concentration












5998400 AOX1

promoter

120572-factorsignal


C-peptide

S S

CN

SSS

S

A chain

B chain

Zeocinresistance

gene

(a)

1 2 3 4 5

257 bpproinsulin

cDNA

(b)


1 2 3 4 5 6 7 8 9 10

14 15 16 17 18 19 20 21 22 23

pPICZ-120572 (36 kb)

257 bpproinsulin

cDNA

257 bpproinsulin

cDNA

(a)

1 2 3 4

257 bpproinsulin

cDNA

(b)



1 2 3 4 5 6 7 8 9 10

257 bpproinsulin

cDNA


5

10

15

25

35

40

55

70

100

130

170kDa

1 2 3 4 5 6 7 8 9 10 M



Proinsulin

1 2 3 4 5 6 7 8 9 10

Commercialinsulin

(a)

Proinsulin

1 2 3 4 5 6 7 8 9 10

(b)










0

20

40

60

80

100

120

140

Glu

cose

conc

(m

gdL

)

Afte

r 7 d

ays o

fin

habi

tatio

n

Afte

r 10

days

of S

TZin

ject

ion

Afte

r 2 h

rs o

f ins

ulin

treat

men

t

Afte

r 4 h

rs o

f ins

ulin

treat

men

t

Afte

r 6 h

rs o

f ins

ulin

treat

men

t






4 Conclusion


Competing Interests




Acknowledgments




References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












5998400 AOX1

promoter

120572-factorsignal


C-peptide

S S

CN

SSS

S

A chain

B chain

Zeocinresistance

gene

(a)

1 2 3 4 5

257 bpproinsulin

cDNA

(b)


1 2 3 4 5 6 7 8 9 10

14 15 16 17 18 19 20 21 22 23

pPICZ-120572 (36 kb)

257 bpproinsulin

cDNA

257 bpproinsulin

cDNA

(a)

1 2 3 4

257 bpproinsulin

cDNA

(b)



1 2 3 4 5 6 7 8 9 10

257 bpproinsulin

cDNA


5

10

15

25

35

40

55

70

100

130

170kDa

1 2 3 4 5 6 7 8 9 10 M



Proinsulin

1 2 3 4 5 6 7 8 9 10

Commercialinsulin

(a)

Proinsulin

1 2 3 4 5 6 7 8 9 10

(b)










0

20

40

60

80

100

120

140

Glu

cose

conc

(m

gdL

)

Afte

r 7 d

ays o

fin

habi

tatio

n

Afte

r 10

days

of S

TZin

ject

ion

Afte

r 2 h

rs o

f ins

ulin

treat

men

t

Afte

r 4 h

rs o

f ins

ulin

treat

men

t

Afte

r 6 h

rs o

f ins

ulin

treat

men

t






4 Conclusion


Competing Interests




Acknowledgments




References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9 10

257 bpproinsulin

cDNA


5

10

15

25

35

40

55

70

100

130

170kDa

1 2 3 4 5 6 7 8 9 10 M



Proinsulin

1 2 3 4 5 6 7 8 9 10

Commercialinsulin

(a)

Proinsulin

1 2 3 4 5 6 7 8 9 10

(b)










0

20

40

60

80

100

120

140

Glu

cose

conc

(m

gdL

)

Afte

r 7 d

ays o

fin

habi

tatio

n

Afte

r 10

days

of S

TZin

ject

ion

Afte

r 2 h

rs o

f ins

ulin

treat

men

t

Afte

r 4 h

rs o

f ins

ulin

treat

men

t

Afte

r 6 h

rs o

f ins

ulin

treat

men

t






4 Conclusion


Competing Interests




Acknowledgments




References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






0

20

40

60

80

100

120

140

Glu

cose

conc

(m

gdL

)

Afte

r 7 d

ays o

fin

habi

tatio

n

Afte

r 10

days

of S

TZin

ject

ion

Afte

r 2 h

rs o

f ins

ulin

treat

men

t

Afte

r 4 h

rs o

f ins

ulin

treat

men

t

Afte

r 6 h

rs o

f ins

ulin

treat

men

t






4 Conclusion


Competing Interests




Acknowledgments




References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article expression and purification of c-peptide

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