Enhanced Production of Recombinant Proteins in Escherichia coli by Filamentation Suppression

During growth of high-cell-density cultures of Escherichia coli,overproduction of recombinant proteins often results in increasedstress response, cell filamentation, and growth cessation. Filamentationof cells consequently lowers final achievable cell concentrationand productivity of the target protein. Reported here is a methodologythat should prove useful for the enhancement of cell growthand protein productivity by the suppression of cell filamentation.By the coexpression of the E. coli ftsA and ftsZ genes, whichencode key proteins in cell division, growth of recombinantstrains as well as production of human leptin and human insulin-likegrowth factor I was improved. Observation of cell morphologyrevealed that the coexpression of the ftsA and ftsZ genes successfullysuppressed filamentation caused by the accumulation of recombinantproteins.

INTRODUCTION

Top

Introduction

Escherichia coli has been the workhorse for the production ofrecombinant proteins because of fast growth, well-known geneticcharacteristics, and the availability of various tools for geneexpression (9). In general, experiments for the overproductionof recombinant proteins begin by the cloning of a gene for thedesired protein into a multicopy plasmid under the control ofa strong inducible promoter. This approach has been successfulin producing large amounts of some proteins but has often resultedin slow growth, increased stress response, and eventually cessationof growth (5, 13). One of the frequent observations during therecombinant protein production in E. coli is that of the filamentationof cells. Cell filamentation causes a reduction in growth rateor no further cell division, which results in low cell concentrationand low productivity of the target proteins.

Cell division in E. coli is a complex process that requirestemporal and spatial coordination of the activities of multiplegene products. At least nine proteins, FtsZ, FtsA, FtsI (PBP3),FtsK, FtsL, FtsN, FtsQ, FtsW, and ZipA, have been shown to beessential components during division process (4). Among these,FtsZ plays a key role, as it initiates cell division by forminga ring-like structure and invaginating the cell wall circumferentiallyat the prospective division site (11). The E. coli mutant lackingthe ftsZ gene was shown to undergo severe filamentation, whichcan be suppressed by the overexpression of the ftsZ gene (1,2). However, it should be noted that overexpression of the ftsZgene can also cause severe cell filamentation or the formationof minicells (14). We also obtained similar results showingthat cells underwent filamentation when the ftsZ gene only wascoexpressed in recombinant E. coli producing human leptin (datanot shown). Some evidence exists that for proper cell division,the fts gene products must be present at appropriate levels(2, 3). In particular, the proper ratio of FtsA to FtsZ (1:100)is required for active cell division. Increase or decrease ofthis ratio can lead to inhibition of the division process (2).We constructed a plasmid, pACfAZ2, that constitutively coexpressesboth the E. coli ftsA and ftsZ genes. Recombinant E. coli strainsoverproducing human leptin and insulin-like growth factor I(IGF-I) were transformed with pACfAZ2 and were subsequentlyexamined for their growth and protein production during high-cell-densitycultivation (HCDC).

Bacterial strains and plasmids.

Top

Bacterial strains and plasmids.

Bacterial strains and plasmids used in this study are listedin Table 1. E. coli XL1-Blue was used as a host strain for thecloning and maintenance of plasmids. E. coli TG1 and W3110 wereused for the production of human leptin and human IGF-I fusionprotein, respectively.

View this table:
[in this window]
[in a new window]
TABLE 1. Bacterial strains and plasmids used in this study

Gene manipulation.

Top

Gene manipulation.

For the construction of a plasmid coexpressing the E. coli ftsAand ftsZ genes, both genes were amplified from E. coli W3110chromosomal DNA by PCR as follows: the forward primer 5'-GGCGGGGATCCTTTTCCTGCTG-3'and the reverse primer 5'-GGGACTGCAGATATTCGATATCACGC-3' weredesigned to contain BamHI and PstI sites, respectively (underlined).The PCR product digested with BamHI and PstI was cloned intopACYC177 to yield pACfAZ2, in which the ftsA and ftsZ genesare expressed from their own constitutive promoters. For theproduction of human leptin, plasmid pTrpAObT was constructedas follows: pEDOb5 was digested with XbaI and EcoRI, and thefragment containing the human obese gene, which encodes thehuman leptin, was cloned into pUC18. The resulting plasmid,p18Ob5, was digested with BamHI and HindIII and was cloned intothe downstream region of the trp promoter in pRL22 to yieldpRLOb5. The SspI-BamHI fragment of pRLOb5 containing the trppromoter and the obese gene was cloned into pTrc99A digestedwith PvuII and BamHI to yield pTrpAObT. For the inducible expressionof human IGF-I fusion protein, plasmid pYKM-I1 was used. Inthis plasmid, the IGF-I gene fused to the end of a truncatedß-galactosidase (163 amino acids) gene is expressedfrom the inducible tac promoter.

Fed-batch culture conditions.

Top

Fed-batch culture conditions.

Growth of fed-batch cultures was carried out in a 6.6-liter-jarfermentor (Bioflo 3000; New Brunswick Scientific Co., Edison,N.J.) at 37°C. Seed culture was prepared in a 1-liter flaskcontaining 200 ml of Luria-Bertani medium (tryptone [10 g/liter],yeast extract [5 g/liter], NaCl [5 g/liter]) at 37°C and250 rpm in a rotary shaker. For the production of leptin inE. coli TG1 the seed culture was transferred into the fermentor,which contained 1.8 liters of R/2 medium (6) plus 20 g of glucose/literand 2 g of yeast extract/liter. Culture pH was maintained at6.8, except for the periods of pH rise following glucose depletion,by the addition of 16.5 M NH₄OH. The dissolved oxygen concentrationwas controlled at 40% of air saturation by automatically increasingthe agitation speed up to 1,000 rpm and by the change of pure-oxygenpercentage. Nutrient feeding solution containing 700 g of glucose/literand 20 g of MgSO₄7H₂O/liter was added according to the pH-stat(with high limit) feeding strategy (9). When the pH rose toa value greater by 0.08 than that of its set point (6.8) dueto the depletion of glucose, the appropriate volume of the feedingsolution was automatically added to increase the glucose concentrationin the culture broth to 0.7 g/liter. Expression of the obesegene was automatically induced by the depletion of tryptophanin the initial medium without the addition of any inducer, suchas 3-ß-indoleacrylic acid. For the production of IGF-Ifusion protein in E. coli W3110, growth of the pH-stat fed-batchcultures was carried out as described above with the followingchanges. Seed culture was transferred into the fermentor, whichcontained 1.8 liters of R/2 medium plus 20 g of glycerol/literand 2 g of yeast extract/liter. Nutrient feeding solution containing500 g of glycerol/liter, 50 g of yeast extract/liter, and 20g of MgSO₄7H₂O/liter was added according to the pH-stat feedingstrategy described above. Expression of the ß-galactosidase-IGF-Ifusion gene was induced at an optical density at 600 nm (OD₆₀₀)of 30 by the addition of isopropyl-ß-D-thiogalactopyranoside(IPTG; Sigma Chemical Co., St. Louis, Mo.) to a concentrationof 1 mM. Ampicillin and kanamycin were added at concentrationsof 50 and 25 mg/liter, respectively, depending on the plasmidsemployed.

Analytical methods.

Top

Analytical methods.

Cell growth was monitored by measuring absorbance at OD₆₀₀.Dry cell weight (DCW) (in grams per liter) was determined asdescribed previously (10). Protein samples were analyzed byelectrophoresis on a sodium dodecyl sulfate-12% (wt/vol) polyacrylamideelectrophoresis gel (8). The protein bands were visualized withCoomassie brilliant blue stain. The contents of leptin and IGF-Iin total protein were quantified with a GS710 calibrated imagingdensitometer (Bio-Rad, Hercules, Calif.), and the total proteinconcentration was determined with a Bio-Rad protein assay kitwith bovine serum albumin as a standard. The concentrationsof leptin and IGF-I produced during growth of the fed-batchcultures were calculated from the total protein concentration,and the content was determined by densitometric scanning. Themorphologies of cells were observed by phase-contrast microscopy(OPTIPHOT-2; Nikon Co., Tokyo, Japan).

Effect of ftsA and ftsZ coexpression on leptin production.

Top

Effect of ftsa and...

First, the effect of coexpression of the ftsA and ftsZ geneson human leptin production was examined. The pH-stat fed-batchcultures of E. coli TG1 harboring pTrpAObT and E. coli TG1 harboringpTrpAObT and pACfAZ2 were carried out. In the fed-batch cultureof E. coli TG1 harboring pTrpAObT (Fig. 1A), the specific growthrate was 0.10 h^-1. The maximum cell concentration of 17.5 gof DCW/liter was obtained in 29 h. Leptin was actively producedfrom 21 h, and the leptin content reached 12.5% of total protein(1.05 g of leptin/liter) at 27 h and then decreased. The volumetricproductivity of leptin was 0.04 g/liter/h. Cells were normalat the beginning of the culture (Fig. 2A) but underwent filamentationafter the accumulation of recombinant leptin (Fig. 2B). In thefed-batch culture of E. coli TG1 harboring pTrpAObT and pACfAZ2(Fig. 1B), the specific growth rate was 0.13 h^-1. The maximumcell concentration of 27.5 g of DCW/liter was obtained in 29h. Active production of human leptin started at 16 h, whichwas earlier than that observed without the coexpression of thefts genes. Leptin concentration and content reached 2.38 g ofleptin/liter and 17.3% of total protein, respectively, at 29h. The volumetric productivity of leptin was 0.08 g/liter/h.Cells maintained normal shape and length even though largerleptin inclusion bodies were accumulated (Fig. 2C and D). Therefore,it can be concluded that the coexpression of the ftsA and ftsZgenes increased both the specific growth rate of recombinantE. coli (1.3-fold) and the volumetric productivity of leptin(2-fold).

View larger version (25K):
[in this window]
[in a new window]
FIG. 1. Time profiles of cell density (OD₆₀₀ [•]), DCW (grams per liter [ ${blacksquare}$ ]), leptin concentration (grams per liter [ ${circ}$ ]), and leptin content in total protein (percent [ ${square}$ ]) during the growth of fed-batch cultures of E. coli TG1 harboring pTrpAObT (A) and E. coli TG1 harboring pTrpAObT and pACfAZ2 (B).

View larger version (75K):
[in this window]
[in a new window]
FIG. 2. Morphologies of recombinant E. coli cells as observed by phase-contrast microscopy. (A and B) E. coli TG1 harboring pTrpAObT; (C and D) E. coli TG1 harboring pTrpAObT and pACfAZ2. Panels A and C depict cells observed at 5 h, while panels B and D depict those observed at 29 h. Black inclusions inside the cells are leptin inclusion bodies.

Effect of ftsA and ftsZ coexpression on IGF-I fusion protein production.

Top

To examine whether this strategy can also be applied to otherexpression and fermentation systems, growth of fed-batch culturesof another recombinant E. coli strain was carried out for theproduction of IGF-I fusion protein under the control of thetac promoter with glycerol as a carbon source. Again, the controlstrain E. coli W3110 harboring pYKM-I1 was compared with E.coli W3110 harboring pYKM-I1 and pACfAZ2. In both cultures,cells were induced with 1 mM IPTG at an OD₆₀₀ of 30, which resultedin the accumulation of IGF-I fusion protein as inclusion bodies.In the fed-batch culture of E. coli W3110 harboring pYKM-I1(Fig. 3A), the specific growth rate was 0.18 h^-1 and the maximumcell concentration was 46.5 g of DCW/liter at 20 h. The IGF-Ifusion protein content reached 10.5% of total protein at 8 hafter induction, and the maximum IGF-I fusion protein concentrationof 2.31 g of IGF-I fusion protein/liter was obtained after 2more hours. The volumetric productivity of IGF-I fusion proteinwas 0.23 g/liter/h. In the fed-batch culture of E. coli W3110harboring pYKM-I1 and pACfAZ2 (Fig. 3B), the specific growthrate was 0.23 h^-1 and the maximum cell concentration was 56.3g of DCW/liter at 20 h. The content and maximum concentrationof IGF-I fusion protein reached 13.5% of the total proteinsand 3.6 g/liter, respectively, at 8 h after induction. The volumetricproductivity of IGF-I fusion protein was 0.45 g/liter/h. Asin the case of the leptin production described earlier, thecoexpression of the ftsA and ftsZ genes increased both the specificgrowth rate (1.3-fold) and volumetric productivity (1.9-fold)of IGF-I fusion protein. In 2 h after induction, the morphologiesof the cells were similar in both cultures (Fig. 4A and C).After the accumulation of IGF-I fusion protein (8 h after induction),cells without the fts coexpression system underwent severe filamentation(Fig. 4B), which was suppressed by the coexpression of the ftsAand ftsZ genes (Fig. 4D). From these results obtained in twoseparate expression and fermentation systems, it can be concludedthat the coexpression of the ftsA and ftsZ genes can suppresscell filamentation during protein overproduction and consequentlyenhance cell growth and protein productivity.

View larger version (25K):
[in this window]
[in a new window]
FIG. 3. Time profiles of cell density (OD₆₀₀ [•]), DCW (grams per liter [ ${blacksquare}$ ]), IGF-I fusion protein (grams per liter [ ${circ}$ ]), and IGF-I fusion protein content in total protein (percent [ ${square}$ ]) during the growth of fed-batch cultures of E. coli W3110 harboring pYKM-I1 (A) and E. coli W3110 harboring pYKM-I1 and pACfAZ2 (B). Vertical dashed lines indicate the time of induction with 1 mM IPTG.

View larger version (90K):
[in this window]
[in a new window]
FIG. 4. Morphologies of recombinant E. coli cells as observed by phase-contrast microscopy. (A and B) E. coli W3110 harboring pYKM-I1; (C and D) E. coli W3110 harboring pYKM-I1 and pACfAZ2. Panels A and C depict cells observed at 1 h after induction, while panels B and D depict those observed at 8 h after induction. Black inclusions inside the cells are inclusion bodies of IGF-I fusion protein.

In conclusion, we report a novel methodology for suppressingcell filamentation that should prove useful for the enhancementof cell growth and productivity of recombinant proteins. Theprimary goal of HCDC is the cost-effective production of desiredproduct by achieving higher volumetric productivity. Reducedgrowth rate and lower protein productivity have been the typicalproblems observed during HCDC. Since most industrial processesfor the production of recombinant proteins involve HCDC, thestrategy of suppressing cell filamentation by the coexpressionof the essential cell division proteins (FtsA and FtsZ) as demonstratedin this paper should prove useful for the enhanced productionof recombinant proteins by HCDC.

ACKNOWLEDGMENTS

This work was supported by the Korean Ministry of Commerce,Industry, and Energy and by the National Research Laboratoryprogram of the Korean Ministry of Science and Technology. Furthersupport from LG Chem Investment Ltd., Samchully PharmaceuticalCo., and BioLeaders Co. is also appreciated. K.J.J. is a postdoctoralfellow supported by the Brain Korea 21 project.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical and Biomolecular Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea. Phone: 82-42-869-3930. Fax: 82-42-869-8800. E-mail: leesy{at}mail.kaist.ac.kr.

REFERENCES

Top