Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures [original]

Kaisa Ukkonen, Johanna Veijola, Antti Vasala, Peter Neubauer

Effect of culture medium, host strain and

oxygen transfer on recombinant Fab

antibody fragment yield and leakage to

medium in shaken E. coli cultures

Article, Published version

This version is available at http://nbn-resolving.de/urn:nbn:de:kobv:83-opus4-68757.

Suggested Citation

Ukkonen, Kaisa ; Veijola, Johanna ; Vasala, Antti ; Neubauer, Peter : Effect of culture medium, host

strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in

shaken E. coli cultures. - In: Microbial Cell Factories. - ISSN 1475-2859 (online). - 12 (2013), art. 73. -

doi:10.1186/1475-2859-12-73.

This work is licensed under a CC BY 2.0 License (Creative

Commons Attribution 2.0 Generic). For more information see

http://creativecommons.org/licenses/by/2.0.

RESEARCH Open Access

Effect of culture medium, host strain and oxygen

transfer on recombinant Fab antibody fragment

yield and leakage to medium in shaken E. coli

cultures

Kaisa Ukkonen

1,2*

, Johanna Veijola

, Antti Vasala

and Peter Neubauer

Abstract

Background: Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct

folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation

parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding

consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and

accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of

media and under different aeration conditions.

Results: Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to

extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like

glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled

shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as

compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from

2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from

intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction

medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤10% to

20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume.

Conclusions: Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration

rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in

autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes

increased Fab accumulation into extracellular medium. These findings have practical implications for screening

applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration

conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency

of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.

Keywords: Fab fragment, Periplasmic expression, Oxygen transfer, Fed-batch, Autoinduction

* Correspondence: [email protected]

Department of Process and Environmental Engineering, Bioprocess

Engineering Laboratory, University of Oulu, Oulu, Finland

BioSilta Oy, Oulu, Finland

Full list of author information is available at the end of the article

Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited.

Ukkonen et al. Microbial Cell Factories 2013, 12:73

http://www.microbialcellfactories.com/content/12/1/73

Background

Fragments of immunoglobulin molecules are widely uti-

lized in therapeutic and diagnostic applications as well

as in basic research. Unlike full-length antibodies, these

smaller fragments, such as the antigen binding frag-

ments (Fab) and single-chain variable fragments (scFv),

are small enough to be produced in Escherichia coli.

However, the yields of correctly folded, functional anti-

body fragments in E. coli are often relatively low and

dependent on the type and primary sequence of the frag-

ment. Yields in the range of 10–20 mg functional Fab

fragments per liter of culture are generally considered

good in shake flask scale [1-3]. Major challenges in bac-

terial antibody fragment expression are the assembly of

separately expressed light and heavy chain to constitute

the functional heterodimer and formation of the four

intra-chain and one inter-chain disulfide bond [4]. Since

the disulfides cannot be efficiently formed in the redu-

cing cytoplasm of E. coli, antibody fragments are most

commonly supplemented with a signal sequence that di-

rects them to the more oxidizing bacterial periplasm for

correct folding. Folded fragments may further leak from

the periplasm into the culture medium, from which

purification can be accomplished without cell lysis [4].

An alternative strategy is to use redox mutant strains

with more oxidizing cytoplasm for folding of the frag-

ments in the E. coli cytoplasm [3,5-7], but these mutant

strains tend to have poor growth that limits their cap-

acity for protein production and scale-up to fermenter

scale.

Previously described approaches to improve antibody

fragment yields in E. coli have mostly focused on the

optimization of the expression construct and the target

fragment itself. For example, co-expression of different

accessory proteins such as the cytoplasmic DnaKJE

chaperone [8] or periplasmic dithiol-disulfide oxidore-

ductases and prolyl cis-trans isomerases [9] have been

reported to increase yields of Fab and scFv fragments. Fu-

sion to maltose-binding protein (MBP) has been shown to

not only increase solubility of antibody fragments [10,11],

but also enhance secretion from periplasm into the cul-

ture medium in secretory E. coli strains [10]. MBP fusion

[12] as well as thioredoxin [13] and SUMO fusions [14]

have also been reported to improve scFv yields in the

cytoplasm of redox mutant strains. In some cases yield

may also be increased by engineering the amino acid se-

quence in non-binding regions of the fragment to reduce

its aggregation tendency [15].

A few reports exist on the optimization of culture

medium and strain selection for antibody fragment pro-

duction. Nadkarni et al. [1] compared defined media

with different carbon sources and induction strategies,

and found Studier’s lactose autoinduction medium to

provide higher Fab yields than either glycerol-based

defined medium with lactose induction or glucose-based

defined medium with IPTG induction. The authors also

compared two expression strains, BL21(DE3) and BL21

(DE3)-RIL, although these strains differ from each other

only regarding rare codon utilization but not regarding

carbon metabolism. The effect of inducer on Fab expres-

sion has also been studied in E. coli K-12 RB791, in

which highest Fab yields were obtained by induction

with either a very low IPTG concentration or 2 g l

-1

lac-

tose using glycerol as the main carbon source [16]. Sup-

plementation of culture medium with L-arginine and

reduced glutathione [17] or sucrose [18] has been de-

scribed as means to increase yields of functional scFvs.

Glutathione was suggested to improve reshuffling of in-

correctly formed disulfides, while the effect of sucrose

was hypothesized to be due to osmotic enlargement of the

periplasmic space and consequently enhanced folding of

the product as a result of reduced local concentration.

Cultivation temperature has been reported to influence

the secretion into the culture medium so that at lower

temperatures the product is more efficiently retained in

the periplasm [18].

In this study we aim to investigate the effects of host

strain, culture medium and aeration conditions on the

production and extracellular leakage of Fab fragments in

shaken E. coli cultures by the example of Fabs binding

specifically to N-terminal pro-brain natriuretic peptide

(NTproBNP), an important diagnostic marker of heart

failure that can be detected from serum by an immuno-

assay applying the anti-NTproBNP Fabs [19]. Three differ-

ent culture media were compared, all of them containing

complex nutrients, but differing in their primary carbon

source as well as in induction strategy. In the Super Broth

medium, peptides, amino acids and sugars of yeast extract

constitute the main carbon source during IPTG-induced

expression. In Studier’s autoinduction medium [20],

growth is first supported by glucose, and when glucose is

exhausted protein expression is autoinduced by diauxic

shift to lactose utilization, while glycerol is also coutilized

as a major carbon source during expression. The third

medium was the fed-batch-like EnBase® medium with

IPTG induction. In this medium the primary carbon

source, glucose, is gradually provided from a soluble poly-

saccharide by biocatalytic degradation [21,22]. The poly-

saccharide used in the current study is different from the

previous reports in that it is also slowly utilized to some

degree through the E. coli maltose-maltodextrin transport

system (own unpublished results). The EnBase fed-batch

-like medium has been successfully used for high-yield

cytoplasmic expression of several non-disulfide bond

containing proteins [22-27] as well as functional protein

with multiple disulfide bonds [28,29], while in this study

we apply this medium for the first time for periplasmic

production of disulfide-containing proteins. We also

Ukkonen et al. Microbial Cell Factories 2013, 12:73 Page 2 of 14

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compared two metabolically different E. coli strains re-

garding their Fab yield in the different growth media.

Apart from differences in Fab yields, we also observed

some peculiar effects on leakage of the Fabs into the cul-

ture medium depending on the type of medium, host

strain, and aeration efficiency.

Results

Comparison of culture media in small scale

Fab fragment expression in E. coli RV308 and E. coli

BL21 was compared in three different media in 24 deep

well plate (24dwp) cultures. Notable differences were ob-

served in both the total yield and localization of the Fabs

(Figure 1). The fed-batch medium provided highest total

yields in both strains, and 60-75% of active product was

found in the extracellular medium at 24 h after induction

(Figure 1 and Table 1). In the autoinduction medium, all

four fragments were produced at high concentrations in

E. coli BL21(DE3), but for three of the fragments the

proportion of extracellular product (40%) was lower

than in the fed-batch medium. Low levels of Fab

activity were detected also in E. coli RV308 when

cultivated in the autoinduction medium, even if this

strain is a Δ(lac)X74 mutant and the expression must

therefore be accounted to leakiness of the promoter.

Fabs were most efficiently transported to extracellular

medium when expressed in the Super Broth medium,

in which 72-97% of product activity was measured in

the extracellular fraction irrespective of fragment or

host strain. However, the total Fab yields in Super

Broth were much lower than in the other two media.

Thus the small scale results suggest that the fed-

batch medium is the most favorable medium for Fab

production due to the high overall yield and efficient

transport of the product to extracellular medium, as

well as the robustness regarding strain type.

The main reason for higher product concentration in

the fed-batch medium compared to the autoinduction

medium appears to be higher cell density (cell density

data for one representative Fab are shown in Table 1) ra-

ther than notably higher productivity per biomass. A re-

liable calculation of product per biomass was however

not possible on the basis of OD

600

, since visual observa-

tion of DNA aggregates in the medium at 42 h indicated

some degree of cell lysis especially in E. coli BL21(DE3)

cultures. Lysis was apparently one of the reasons for Fab

release from periplasm to medium in E. coli BL21(DE3),

and possibly also in E. coli RV308.

F32

100

120

140

Fab concentration [mg l

-1

]

100

120

140

Periplasm

Medium

F16

RV308EB

RV308ZYM

RV308SB

BL21EB

BL21ZYM

BL21SB

Fab concentration [mg l

-1

]

100

120

140

160

180

200

1B10

RV308EB

RV308 ZYM

RV308SB

BL21EB

BL21ZYM

BL21 SB

Figure 1 Yields of Fab fragments in mg per liter of culture in different media. Quantities of Fab fragments F1, F16, F32 and 1B10 were

measured by antigen-binding ELISA from cell lysate (periplasmic fraction; in black) and broth supernatant (medium fraction; in grey).

All fragments were expressed in E. coli RV308 and BL21 in 24 deep well plates. Samples were drawn for analysis at 24 h after induction in the

fed-batch-like EnBase medium (EB), 19 h after induction in Super Broth (SB), and 19 h and 42 h after cultivation start in ZYM-5052 autoinduction

medium (ZYM; 19 h data not shown, yields at 19 h were lower than at 42 h). The mean values of two independent replicate cultivations

are shown.

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Cultivation in Super Broth resulted in high final pH

ranging from 8.0 to 8.5 (data for one representative

Fab are shown in Table 1), while in the fed-batch and

autoinduction media pH remained at a lower and more

neutral range (6.6-7.2 depending on the clone and

medium, except for the clones expressing Fab 1B10 which

resulted in pH decrease to levels below 6.0; data not

shown). The pH increase in Super Broth is in line with our

earlier observations on pH development in complex media

without added monosaccharide carbon sources [22,23],

and likely limited both the final cell density and Fab yield.

Medium composition, respiratory activity and Fab

localization

A separate small-scale cultivation was performed to

study the influence of fed-batch medium composition

on the dynamics of dissolved oxygen tension (DOT) dur-

ing Fab fragment expression in E. coli RV308 (Figure 2).

The pre-induction medium composition was kept con-

stant, and modification was achieved by addition of

more nutrients at the time of induction. Switch from ini-

tially unlimited growth to fed-batch-like limited growth

took place at 9–10 h, and DOT at the time of induction

(18 h) was 80-100% in all cultures. The cultures that did

not receive complex nutrient supplementation and more

glucose-releasing biocatalyst at induction maintained

DOT at 100% after induction (Figure 2a). Addition of

complex nutrients and more biocatalyst at induction (18

h) resulted in increased respiratory activity, and conse-

quently DOT remained at a lower level (20-30%) for a

period of 8–10 h after induction (Figure 2b). The in-

creased oxygen consumption by addition of complex nu-

trients and increased glucose release was associated with

high Fab activity in the extracellular medium (66-73% of

total Fab activity, Figure 2b; see also in Additional file 1:

Table S1b), while in the cultures with lower respiration

and 100% oxygen saturation the product remained

mostly in the periplasm (Figure 2a; see also in Additional

file 1: Table S1a). pH was maintained between 7.0 and

7.5 in both cases (data not shown). Though the inde-

pendent effects of DOT, growth rate and metabolic

changes on Fab localization cannot be evaluated separ-

ately in this experiment, the results demonstrate that in

the fed-batch medium the ratio of periplasmic and extra-

cellular Fab can be drastically changed by modifying the

availability of carbon and nitrogen substrates and conse-

quently the respiratory rate after induction.

Influence of shaking speed on Fab yield and localization

Expression of the Fab fragments in shake flask scale

demonstrated that the yield and extracellular leakage

can be influenced by modification of aeration efficiency

via shaking speed. Cultures in the fed-batch medium were

incubated at 250 rpm shaking speed in baffled Ultra Yield

Flasks™(UYF) up until induction, after which the speed

was either reduced to 150 rpm (providing k

a~200 h

-1

[30]) or kept at 250 rpm (providing k

a~500 h

-1

[23]). Ex-

pression in E. coli RV308 at the lower shaking speed

resulted consistently in higher yields of fragments F1, F16

and F32, even if there was some experiment-to-experi-

ment variation in yield between replicates (Figure 3). Re-

duction of shaking speed also resulted in significant

changes in Fab localization so that most of the Fab activity

was detected in the medium as opposed to the efficient

periplasmic retention of Fab at 250 rpm (Figure 3). This

effect was observed for F1 and F32 in two out of three

replicate experiments (A and C in Figure 3) at 150 rpm,

while in the third experiment (B) there was much less

leakage into the medium. Despite this inconsistency,

which is may be caused by differences in oxygen uptake

rate (OUR) between the replicates, the data suggest that

there is a tendency towards higher extracellular Fab

accumulation under conditions of lower oxygen supply.

The extracellular proportion of fragment F16 was lower

than for the other fragments, but consistently higher at

150 rpm compared to 250 rpm. Unlike the other three

fragments, 1B10 leaked efficiently into the medium

already at 250 rpm (data for 1B10 is shown in Additional

Table 1 Cell density, pH and percentage of extracellular Fab in different media in 24dwp

% of Fab

in medium

600

19 h 24 h 42 h 19 h 42 h

RV308 EnBase 65.3 ± 2.6 21.7 ± 1.9 26.9 ± 2.7 7.07 ± 0.01

ZYM-5052 6.8 ± 1.6 12.4 ± 3.7 14.1 ± 0.8 6.90 ± 0.02

Super Broth 96.1 ± 2.4 13.8 ± 0.5 8.31 ± 0.18

BL21(DE3) EnBase 69.6 ± 2.6 19.8 ± 1.8 21.7 ± 6.5 6.70 ± 0.06

ZYM-5052 39.8 ± 15.2 16.8 ± 5.1 17.5 ± 7.5 7.15 ± 0.02

Super Broth 93.1 ± 1.9 8.5 ± 0.5 8.5 ± 0.04

Cell density was determined by optical density measurements at 600 nm (OD

600

), and pH was measured from culture supernatant at room temperature. Mean

and standard deviation of two independent replicate experiments are shown. In the fed-batch-like cultures (EnBase) OD

600

is shown at 24 h (6 h from induction)

and 42 h (25 h from induction). In ZYM-5052 autoinduction cultures OD

600

is shown at 19 h and 42 h from cultivation start. In Super Broth cultures OD

600

was

measured after 19 h incubation in the inducing medium. pH was measured at the end of cultivation. The percentage of extracellular Fab represents the situation

at the end of cultivation.

Ukkonen et al. Microbial Cell Factories 2013, 12:73 Page 4 of 14

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file 2: Table S2a), and hence no difference in leakage was

observed at different shaking speeds.

The degree of cell lysis was estimated by total protein

measurement from cell pellet and culture supernatant by

Bradford assay. Comparison of the percentage of cell

lysis (as estimated from the relative concentrations of

total protein in the cell pellet and in the medium; see in

Additional file 2: Table S2a for the lysis estimates) and

the percentage of Fab found in the culture medium sug-

gests that at 250 rpm the small amount of fragments F1,

F16 and F32 detected in the medium was released by

cell lysis and there was no notable leakage from intact

cells. The higher extracellular Fab yield at 150 rpm was

partly due to higher cell lysis, but as the percentage of

lysis was much lower than the percentage of extracellu-

lar Fab it is apparent that there was also increased leak-

age from intact cells. Depending on the fragment, at

least 20-40% of total functional Fab leaked into the

medium without accompanying lysis at 150 rpm. The

possibility that the reduction of extracellular Fab fraction

at the higher shaking speed might result from Fab de-

naturation due to the very efficient and turbulent shaking

was ruled out by demonstrating over 95% preservation of

binding activity when Fab-containing cell-free broth was

shaken at 250 rpm for 24 h (data not shown).

Similar effect of shaking speed on yield and localization

was observed for E. coli BL21(DE3) in the autoinduction

medium, when cultures were performed in the UYF bot-

tles with either 150 or 250 rpm shaking speed from the

beginning. Total yields of F1, F16 and F32 were much

higher at 150 rpm, and leakage of Fab into the medium

also increased significantly at the lower shaking speed

(Figure 4). The degree of lysis was low at both shaking

speeds, but percentage of extracellular Fab increased from

≤10% to 20-30% of total Fab activity when the speed was

reduced from 250 to 150 rpm (see in Additional file 2:

Table S2b for the lysis estimates and percentages of extra-

cellular Fab). Total yield of 1B10 in the autoinduction

medium was not affected by the shaking speed (Figure 4),

but extracellular Fab activity increased from 3 to 88%

when speed was reduced to 150 rpm.

E. coli BL21(DE3) cultures in the fed-batch medium

released Fabs very efficiently into the medium so that ir-

respective of shaking speed 87-97% of total Fab activity

was detected in the medium after 24 h expression period

(Figure 4; see also in Additional file 2: Table S2c for the

0.3 U/l 0.6 U/l 1.5 U/l

Fab concentration

[mg l

-1

]

100

150

200

Time [h]

10 20 30 40

DOT %

100

120

140

Time [h]

0 10203040

DOT %

100

120

140

0.3 U/l 0.6 U/l 1.5 U/l

Fab concentration

-1

]

100

150

200

Periplasm

Medium

Figure 2 Influence of fed-batch medium composition on oxygen saturation and Fab production. Dissolved oxygen tension (DOT, % of

saturation) was measured online during 24 microwell plate expression of Fab fragment F1 in E. coli RV308 in the fed-batch-like EnBase medium.

Fab yields in mg per liter of culture were measured by antigen-binding ELISA from cell lysate (periplasmic fraction; in black) and broth

supernatant (medium fraction; in grey) at 41 h. a: Cultivation without addition of nutrients at induction; b: Cultivation with addition of complex

nutrients and 3 U l

-1

biocatalyst at induction (18 h, indicated by arrows). In each case, Fab yields are shown for cultures with initial biocatalyst

concentrations of 0.3, 0.6 and 1.5 U l

-1

. Representative DOT graphs are shown from the cultures with initially 0.6 U l

-1

biocatalyst. DOT profiles

with initial biocatalyst concentrations of 0.3 and 1.5 U l

-1

were essentially similar to the graphs shown. Standard deviations for the ELISA analysis

are shown in Additional file 1.

Ukkonen et al. Microbial Cell Factories 2013, 12:73 Page 5 of 14

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percentages). Cell lysis was also substantial, typically

40-50% (lysis estimates are shown in Additional file 2:

Table S2c). Total Fab yields were higher at the lower

shaking speed, but the effect was less prominent than

in the autoinduction medium.

Based on measurements at a few selected time points,

pH was not significantly affected by the shaking speed in

E. coli RV308 cultures (pH data are shown in Additional

file 3: Tables S3a-S3c), and the differences in Fab yield

and leakage are therefore not likely to be due to pH

changes. In E. coli BL21(DE3), pH in the fed-batch

medium was lower at the lower shaking speed, while in

the autoinduction medium lower shaking speed contrib-

uted to consistently ~0.4 units higher pH. The pH

change in fed-batch medium had apparently no influ-

ence on the extracellular Fab ratio in E. coli BL21(DE3).

Influence of culture volume on Fab yield and localization

The finding that a change in shaking speed could so

drastically influence Fab localization was unexpected,

and we wanted to see whether this effect could be

reproduced by modification of aeration efficiency via the

culture surface to volume ratio. This was studied by

varying the culture volume between 1 and 5 ml in the

wells of a 24dwp. The results with Fab F1 expressed in

E. coli RV308 in the fed-batch medium demonstrated

significantly increased leakage into the extracellular

medium with increasing culture volume (Figure 5a). The

threshold was between 3 ml and 4 ml so that at 3 ml

92% of total Fab activity was retained in the periplasm,

while at 4 ml 66% of Fab activity was found in the cul-

ture medium (the percentages of extracellular Fab are

shown in Additional file 4: Table S4). When culture vol-

ume was further increased to 5 ml the total yield was re-

duced and anaerobic metabolism was indicated by a low

pH (Figure 5b). These results with E. coli RV308 were

reproduced in an independent repetition of the experi-

ment. The data demonstrate that Fab localization may

be drastically changed by a relatively small change in

aeration efficiency, such as increase of culture volume by

one third.

In E. coli BL21(DE3) cultures in the autoinduction

medium the influence of culture volume on total Fab

yield was minor (Figure 5a). It is likely that even at the

lowest volume (1 ml) oxygen supply was below the

threshold that caused significant productivity loss in the

shake flask cultures at high shaking speed. Increasing

culture volume contributed to gradual increase in the

extracellular Fab fraction from 8% in 1 ml culture up to

28% in 5 ml culture (Figure 5a; see also in Additional file

4: Table S4 for the percentages of extracellular Fab). As

E. coli cannot grow anaerobically on glycerol, no acidifi-

cation was observed in the autoinduction medium with

increasing severity of oxygen limitation (Figure 5b).

F32

250rpmA

250rpmB

250 rpmC

150rpmA

150rpmB

150 rpmC

Fab concentration [mg l-1]

100

120

F16

250rpmA

250rpmB

150rpmA

150rpmB

Fab concentration [mg l

-1

]

100

150

200

250

250rpmA

250rpmB

150rpmA

150rpmB

150rpmC

Fab concentration [mg l-1]

100

120

140

160

Periplasm

Medium

Figure 3 Fab expression in E. coli RV308 in shake flask cultures.

Yields in mg per liter of culture for Fab fragments F1 (a), F16 (b) and

F32 (c) expressed in E. coli RV308 in the fed-batch-like EnBase

medium in Ultra Yield shake flasks at two different shaking speeds

(150 rpm and 250 rpm), as measured by antigen-binding ELISA from

cell lysate (periplasmic fraction; in black) and broth supernatant

(medium fraction; in grey). Samples were drawn 24 after induction.

A-C on the horizontal axis refer to independent replicate

experiments. Standard deviations for the ELISA analysis are shown in

Additional file 2.

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Timeline of Fab leakage

To get a more detailed insight into the Fab release from

periplasm to medium and the role of cell lysis in this,

Fab accumulation and OD

600

profiles were recorded

from 150 rpm shake flask cultures in the fed-batch

medium with both expression strains. Fragment F1 was

expressed as the representative fragment. Fab accumula-

tion into the medium started at approximately 9 h

and 5 h after induction in E. coli RV308 and E. coli

BL21(DE3), respectively (Figure 6). At the same time,

Fab activity in the periplasm and OD

600

were both

still increasing, which indicates that the culture was

not yet in stationary phase and not susceptible to cell

lysis. At 14 h after induction, the proportion of extra-

cellular Fab of total Fab activity at the time was 30% in

RV308 and 50% in BL21(DE3), which can be accounted to

lysis-independent leakage. When cultivation was contin-

ued into stationary phase (past 14 h from induction), part

of the cells lysed and released more Fab into the medium,

as indicated by a reduction in OD

600

. In the end, 75% and

92% of total Fab activity was found in the medium in

RV308 and BL21(DE3), respectively. Based on OD

600

the degree of lysis between 14 h and 25 h was 25%

in RV308 and 46% in BL21(DE3). However, decrease

in OD

600

may also be partly due to shrinkage of cell

size as the cells switch from active growth phase to

stationary phase [31], and hence the degree of lysis

may be slightly overestimated from the OD

600

data.

Assuming 25% lysis in RV308 after 14 h, the maximum

amount of Fab released by lysis is 0.25 × (total Fab activity

at 25 h –extracellular Fab activity at 14 h). Hence it is cal-

culated that during the 25 h expression period at least

55% of total active Fab leaked into the medium without

accompanying cell lysis. Correspondingly, the percentage

of Fab leakage without lysis is estimated to be at least 65%

of total Fab in BL21(DE3). The data demonstrate that Fab

leakage in the fed-batch medium begins several hours

before significant lysis, and thus it is possible to harvest

extracellular Fab in the absence of cytoplasmic proteins by

optimizing the harvest time.

Combined with the data on oxygen consumption

(Figure 2), though from a different cultivation, the

pattern of Fab accumulation (Figure 6) suggests that

Fab concentration [mg l-1]

100

Periplasm

Medium

F16

Fab concentration [mg l-1]

100

F32

Fab concentration [mg l-1]

1B10

EB250rpm

EB150rpm

ZYM250rpm24h

ZYM250rpm41h

ZYM150rpm24h

ZYM150rpm41h

Fab concentration [mg l-1]

Figure 4 Fab expression in E. coli BL21(DE3) in shake flask

cultures. Yields in mg per liter of culture for Fab fragments F1 (a),

F16 (b), F32 (c) and 1B10 (d) expressed in E. coli BL21(DE3) in Ultra

Yield shake flasks at two different shaking speeds (150 rpm and 250

rpm), as measured by antigen-binding ELISA from cell lysate

(periplasmic fraction; in black) and broth supernatant (medium

fraction; in grey). All fragments were expressed in the fed-batch-like

EnBase medium (EB) and ZYM-5052 autoinduction medium (ZYM).

Samples were drawn for analysis at 24 h after induction in EB, and

24 h and 41 h after cultivation start in ZYM. Standard deviations for

the ELISA analysis are shown in Additional file 2.

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the leakage of Fab in E. coli RV308 starts around the

time when respiratory activity of the culture decreases

and oxygen level increases. DOT recording in small-

scale E. coli BL21(DE3) expression culture (data not

shown) indicated that in this strain a similar decrease

in oxygen consumption takes place at 4–5hafterin-

duction, which also coincides the start of Fab accu-

mulation into the medium.

Discussion

The finding that total Fab yields were reduced by high

aeration was unexpected and contradictory to our earlier

results with cytoplasmically expressed recombinant pro-

teins in the fed-batch medium [23]. The largest effect

of aeration on total Fab yield was observed in the

autoinduction medium, in which total yield increased

by 5 to 9-fold when shaker speed was reduced from

250 to 150 rpm. This is consistent with an earlier re-

port by Blommel et al. [32], who demonstrated that

protein expression in autoinduction media is highly

dependent on the oxygenation state of the culture so

that under oxygen limited conditions lactose con-

sumption is preferred over consumption of glycerol,

which in turn promotes earlier induction and higher total

yield of the recombinant protein. The sensitivity of lactose

and glycerol utilization patterns to oxygen availability

could explain the adverse effect of high aeration on Fab

expression in the autoinduction medium, but the reason

for yield reduction under high aeration in the fed-batch

medium is not clear. It is known that increased DOT can

cause oxidative damage to recombinant proteins and their

expression [33], but further studies would be needed to

elucidate whether the observed reduction in functional

Fab yield is due to oxygenation-dependent changes in the

host metabolism or in the oxidative folding of Fab frag-

ments in the periplasm. Interestingly, it seems that high

DOT might be less detrimental to Fab expression in the

fed-batch medium when the complex nutrient supple-

mentation at induction is excluded and the post-induction

growth rate is lower.

It is commonly acknowledged that antibody fragments

can leak from E. coli periplasm to culture medium [4,34],

and that this leakage takes place especially during

extended cultivation periods [35]. In this study we ob-

served the accumulation of Fab fragments in extracellular

medium to increase under conditions of lower oxygen

BL21(DE3) ZYM

Culture volume [ml]

12345

RV308 EnBase

Culture volume [ml]

12345

Fab concentration [mg l-1]

100

120

140

160

Periplasm

Medium

RV308 EnBase

Culture volume [ml]

12345

5,5

6,0

6,5

7,0

7,5

8,0

bBL21(DE3) ZYM

Culture volume [ml]

12345

Figure 5 Influence of culture volume in 24 deep well plates. Yields of Fab fragment F1 in mg per liter of culture (a) and culture pH (b) in 24

deep well plate cultivations with varying broth volume (1–5 ml). Fab quantities were measured by antigen-binding ELISA from cell lysate

(periplasmic fraction; in black) and broth supernatant (medium fraction; in grey). Fab was expressed in E. coli RV308 in the fed-batch-like EnBase

medium, and in E. coli BL21(DE3) in ZYM-5052 autoinduction medium (ZYM). Samples were drawn for analysis at 24 h after induction in EnBase

and 42 h after cultivation start in ZYM. Standard deviations for the ELISA analysis are shown in Additional file 4.

Ukkonen et al. Microbial Cell Factories 2013, 12:73 Page 8 of 14

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availability. This effect was observed in E. coli RV308 in

the fed-batch medium, and in E. coli BL21(DE3) in the

autoinduction medium. Part of the increased release of

Fab into the cultivation medium can be accounted to in-

creased cell lysis, but also leakage without lysis appears to

increase significantly when aeration efficiency is reduced.

The increase in Fab leakage could be due to the dir-

ect influence of DOT during expression, or due to

changes in growth rate as a result of reduced oxygen

supply. Growth rate has been reported to modify the

outer membrane protein and lipid composition, and

consequently influence the efficiency of periplasmic pro-

tein leakage [36,37]. In the study of Bäcklund et al. [37],

increased growth rate in glucose-limited fed-batch con-

tributed to higher product leakage into the extracellular

medium, while according to Shokri et al. [36] the influ-

ence of growth rate may not be linear as they observed

maximum leakage at a growth rate of 0.3 h

-1

, below or

above which leakage decreased significantly. Both cell lysis

and leakage from intact cells were influenced by the

growth rate, and these were accompanied by changes in

outer membrane lipid composition so that maximum in

unsaturated fatty acids and minimum in saturated fatty

acids coincided with the maximum in protein leakage.

Therefore, changes in growth rate may be at least part of

the mechanism by which the modification of oxygen sup-

ply via shaking speed or surface to volume ratio influenced

the Fab leakage in our study. Growth rate during Fab ex-

pression could also be a contributing factor to the differ-

ences in the ratio of periplasmic and extracellular Fab

observed between the different growth media.

Aeration can also influence the membrane lipid com-

position independent of growth rate, as has been earlier

shown in chemostat cultures [38]. Decrease in aeration

rate was reported to result in a decrease in unsaturated

fatty acids and increase in cyclopropane fatty acids. An

earlier study also reported similar changes in response

to lower aeration rate [39]. The effect of these changes

on protein leakage was not studied, but since both the

decrease in unsaturated fatty acids and the increase in

cyclopropane acids are known to reduce membrane

fluidity they can be expected to contribute to reduced

protein leakage. This seems contradictory to our findings

that showed increased leakage at lower aeration rate

even when the effect of lysis was subtracted. On the

other hand, our data suggest that the beginning Fab

leakage may coincide with an increase in DOT after a

period of low oxygen saturation. Such a sharp change in

DOT level contributes to substantial changes in the rela-

tive abundance of several outer membrane proteins [40],

and this reorganization of the membrane structure could

promote higher membrane permeability and leakage of

the periplasmic product. Alternatively, it could also be

the cumulative accumulation of Fab in the periplasm

that eventually initiates leakage due to diffusive pressure,

and decrease in OUR could coincide this moment as a

result of the stress of high Fab accumulation on the cell

and consequent decrease in growth rate. Since reduced

aeration generally contributed to higher total yield of Fab,

the diffusive pressure would be higher under these condi-

tions. Also the increased transport of recombinant product

to periplasm might in itself reduce the ability of the cell to

transport structural elements to the outer membrane [36],

resulting in a more permeable membrane structure

allowing for higher diffusive leakage after sufficient product

accumulation in the periplasm. However, total Fab concen-

tration and leakage were not always correlated. In some

cases increased leakage was observed without accompany-

ing increase in total yield, suggesting that the leakage is

more dependent on other factors than the periplasmic Fab

concentration. These most likely include changes in the

RV308

0 5 10 15 20 25 30

Fab, % of total

100

600

Fab in periplasm

Fab in medium

600

BL21(DE3)

Time from induction [h]

0 5 10 15 20 25 30

Fab, % of total

100

600

Figure 6 Dynamics of Fab accumulation into periplasm and

extracellular medium. Fab fragment F1 was expressed in E. coli

RV308 and BL21(DE3) in the fed-batch medium and Ultra Yield

shake flasks at 150 rpm shaking. Fab quantities in cell lysate

(periplasmic fraction; solid line with black circle) and broth

supernatant (medium fraction; dashed line with triangle) were

measured by antigen-binding ELISA, and are shown at different time

points as percentage of the final Fab yield (left vertical axis). Cell

density was recorded by OD

600

readings (dotted line with square),

which are shown on the right vertical axis.

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outer membrane composition due to either direct or indir-

ect effects of DOT.

Extracellular pH may also affect the membrane fatty

acid composition and hence the leakage efficiency of

periplasmic proteins. There seems to be a tendency to-

wards higher percentage of unsaturated fatty acids and

lower percentage of cyclopropane acids with increasing

pH [39], which suggests that higher pH might promote

higher membrane permeability. However, we observed

that lower oxygen availability contributed to increased Fab

leakage in E. coli RV308 also in the absence of notable pH

change, while in E. coli BL21(DE3) a pH decrease caused

by reduced oxygen supply in the fed-batch medium was

not associated with changes in Fab localization. Moreover,

reduced aeration efficiency had opposite effects on pH in

the fed-batch and autoinduction media, whereas Fab leak-

age increased in both. Thus it seems that the effect of pH

at least in the range of 6.4 to 7.4 is minor, if any, and aer-

ation influences Fab leakage by other mechanisms.

While further studies would be needed to confirm the

independent effects of DOT, growth rate and pH on Fab

leakage, our findings about the changes in Fab yield and

leakage in response to aeration efficiency and medium

composition have important practical implications for

Fab production in shaken cultures. It is usually most

straightforward to purify Fab fragments directly from the

culture medium, and hence the goal in Fab production is

in most cases to maximize the extracellular yield. Based

on our results the extracellular Fab yield can be maxi-

mized by cultivation in the fed-batch medium with com-

plex nutrient supplementation under moderately oxygen

limited conditions. Both E. coli BL21 and E. coli RV308

are good hosts for the extracellular production in the

fed-batch medium. However, maximum Fab accumula-

tion in the culture medium requires long cultivation pe-

riods during which cell lysis takes place to a significant

degree, resulting in presence of background cellular pro-

teins in the medium. In this regard E. coli RV308 seems

to be a convenient strain for production of extracellular

antibody fragments, as it has lower lysis rate than E. coli

BL21 but under sufficiently oxygen-limited conditions

can release substantial amounts of Fab into the cultiva-

tion medium in a lysis-independent manner. In both

strains, however, maximum recovery of extracellular

product while minimizing release of cytoplasmic pro-

teins is a matter of optimizing the harvest time. In some

specific cases it may be preferable to collect Fabs from

the periplasm, and the best strategy for maximizing the

periplasmic yield seems to be expression in E. coli

RV308 and the fed-batch medium with exclusion of the

complex nutrient supplementation at induction. This ap-

proach minimizes Fab leakage and maintains higher overall

yield than cultivation with the nutrient supplementation

under high aeration conditions. Since maximum Fab

expression is achieved at relatively low aeration rates, Fab

production at larger scale could be well accomplished in

vessels such as disposable wave-mixed bioreactors [41] that

have lower aeration rates compared to stirred bioreactors.

The enzyme-based fed-batch system should be well suited

to larger scale Fab production as it has been demonstrated

well scalable up to pilot plant scale [42] and applicable to

disposable bag bioreactors [43].

Our results also highlight the importance of aeration

rate as a cultivation parameter in laboratory-scale shaken

cultures which are often performed without appropriate

consideration of oxygen transfer. If the aeration effi-

ciency and other factors contributing to oxygen satur-

ation during cultivation are not controlled when the

system is scaled up from one type or size of vessel to an-

other, productivity of the culture may vary considerably

due to changes in DOT. Changes in aeration can also re-

sult in surprising effects beyond expression yield, as was

the case with periplasmic protein leakage in this study.

Conclusions

In conclusion, we demonstrated that the yield and leakage

of Fab fragments are highly dependent on expression

strain, culture medium, aeration efficiency, and the com-

bination of these parameters. High yields of Fab fragments

were obtained in both E. coli K-12 strain and BL21 strain

in a medium with fed-batch-like glucose feeding, and

in E. coli BL21 in a glycerol-based autoinduction

medium. Regardless of strain and medium, maximum

volumetric productivity was achieved under sufficiently

oxygen-limited conditions. Also the leakage of Fabs into

the culture medium increased considerably under lower

aeration conditions. This dependency may cause gaps in

reproducibility when scaling up or down if oxygen supply

or consumption rate are changed, but it also offers a prac-

tical way to efficiently manipulate the ratio of product

localization in periplasm and extracellular medium.

Materials and methods

Expression constructs

The gene sequences encoding four different Fab frag-

ments (Veijola et al., manuscript in preparation) against

N-terminal prohormone of brain natriuretic peptide

(NTproBNP) were each cloned to a modified pKK233

expression vector backbone (Veijola et al., manuscript in

preparation) under the control of tac promoter, and

transformed into E. coli BL21(DE3) and RV308 using

standard cloning and transformation procedures. The

pKK233 vector encodes resistance to ampicillin. The Fab

fragments contained an N-terminal pelB signal sequence

for periplasmic transport in both the heavy and the light

chains. Additionally, a C-terminal hexahistidine tag was

included in the heavy chain. Three of the Fab fragments

(coded as F1, F16 and F32) bind to their epitopes near

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the C-terminal end of NTproBNP, while one fragment

(coded as 1B10) binds near the N-terminal end of the

antigen. The general layout of the expression construct

is shown in Figure 7.

Media

Fed-batch-like cultivation conditions were provided by

using the EnBase system with enzyme-based glucose re-

lease from soluble polysaccharide. The medium was pre-

pared by dissolving EnPresso® medium tablets (BioSilta,

Oulu, Finland) into sterile water. As described previously

[22], the medium consists of mineral salts, MgSO

thiamine, trace elements solution, soluble polysaccharide

substrate, and a low amount of complex nutrients. After

dissolution of the tablets, the medium was supplemented

with 1 g l

-1

glucose and pH was adjusted to 7.4 by

adding 1.6 ml of 2M NaOH to each 100 ml of medium.

Cultures in shake flasks were supplied with 0.6 U l

-1

the glucose-releasing biocatalyst (EnZ I’m, BioSilta)

before inoculation. Screening cultures in 24 deep well

plates were grown as a batch without biocatalyst until

induction. At the time of induction, all cultures in 24

deep well plates and shake flasks were supplied with 3 U

-1

biocatalyst and the EnPresso Booster (BioSilta) pro-

viding complex nutrients (peptone and yeast extract).

Super Broth medium with MOPS buffering (SB-MOPS)

contained (per liter): tryptone 35 g, yeast extract 20 g,

NaCl 5 g, MOPS 10 g; pH was adjusted to 7.0. SB-MOPS

for pre-induction growth was supplemented with 2 g l

-1

glucose, and for induction the cells were transferred to

fresh glucose-free SB-MOPS.

ZYM-5052 autoinduction medium [20] contained

(per liter): tryptone 10 g, yeast extract 5 g, Na

HPO

3.56 g, KH

3.40 g, NH

Cl 2.68 g, Na

0.71 g, gly-

cerol 4 ml, glucose 0.5 g, lactose 2 g, trace elements solu-

tion 2 ml, and MgSO

3mM.

All media contained 100 μgml

-1

ampicillin for selective

maintenance of the plasmid. For cultivation in the baffled

shake flasks media were supplemented with 0.1 ml l

-1

antifoam (Sigma 204).

Deep well plate cultivations

Culture media were inoculated with Fab–expressing

clones with high cell density glycerol stocks (OD

600

30–70) to OD

600

of 0.1-0.15. Broth volume was 3 ml in

round-bottom square-shaped wells of 24-deep well plates

(24dwp; Thomson Instrument, Part No. 931565-G-1X),

and the plates were covered with adhesive porous mem-

brane seals (Thomson Instrument, Part No. 899410).

All plate cultivations were performed at 250 rpm in

an orbital shaker with 25 mm offset (Infors HT Multitron,

Infors AG). Under these conditions, the approximate

evaporation rate was 7% of original volume within 19 h

and 23% within 42 h. The concentration of broth as a

result of evaporation as well as the dilution of the

fed-batch cultures due to Booster addition were both

accounted for when calculating the results so that the

evaporation and dilution effects were eliminated from

the Fab concentrations.

Cultures in the fed-batch medium were grown over-

night at 30°C, followed by induction at 17 h with 0.2

mM IPTG and simultaneous addition of 10x Booster

concentrate (to 1:10 v/v) and biocatalyst (3 U l

-1

). Incu-

bation was continued for further 24 h at 30°C.

Cultures in Super Broth medium were grown with 2 g

-1

glucose to OD

600

of 0.5-0.8. 2 × 3 ml cultures were

then collected into a single vial, and cells were gently

spun down at room temperature. Supernatant was

discarded and the pellet was resuspended in 3 ml of

glucose-free SB-MOPS with 0.05 mM IPTG (for E. coli

RV308) or 0.2 mM IPTG (for E. coli BL21). IPTG con-

centrations had been previously optimized for maximum

Fab expression in SB-MOPS (data not shown). The sus-

pension was transferred back to 24dwp and incubated

for 19 h at 30°C.

Autoinduction cultures in ZYM-5052 medium were

incubated for 41 h at 30°C.

Shake flask cultivations

For flask-scale expression, 50 ml cultures were inocu-

lated with high cell density glycerol stocks to OD

600

0.1-0.15 and incubated in 250 ml baffled Ultra Yield

Flasks (UYF; Thomson Instrument, Part No. 931144)

covered with adhesive airporous membranes (AirOtop;

Thomson Instrument, Part No. 899423). Temperature

was 30°C for all flask experiments, and the offset of or-

bital shaking was 25 mm.

Cultures in fed-batch medium were grown at 250 rpm

with 0.6 U l

-1

of the glucose-releasing biocatalyst for

17 h, and then induced with 0.2 mM IPTG. Together

pelB VLCL

Ptac

EcoRI

NheI

AscI

pelB VHCH6xHis

SfiI

NotI

HindIII

SD SD

Figure 7 Schematic presentation of the Fab expression construct. The Fab fragment is arranged as a bicistronic unit with the light chain

) and heavy chain (V

) in different reading frames. Each chain is equipped with pelB signal sequence in the N-terminus and the heavy

chain is fused with C-terminal hexahistidine tag (6xHis). Ptac: tac promoter, SD: Shine-Dalgarno ribosome binding site.

Ukkonen et al. Microbial Cell Factories 2013, 12:73 Page 11 of 14

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with IPTG, one EnPresso Booster tablet and 3 U l

-1

of the biocatalyst were added. Shaking speed after in-

duction was alternatively 250 rpm or 150 rpm.

Cultures in autoinduction medium were incubated at

250 rpm or alternatively at 150 rpm.

In all shake flask experiments, broth volume was mea-

sured every time a sample was taken. This data was used

in the calculation of results to eliminate the effect of dif-

ferent evaporation rates from Fab concentrations and

OD data.

Cultivation with online oxygen monitoring

An additional experiment was performed with the

fed-batch medium in a 24 round-well plate with inte-

grated optical oxygen sensors (OxoDish®, PreSens GmbH,

Regensburg, Germany) in each well. The plate was placed

onto SDR SensorDish® Reader (Presens GmbH), and the

plate and reader were fixed to an orbital shaker with

50 mm offset. Cultivations were performed with 1.1.

ml culture volume at 30°C and 200 rpm with online

recording of dissolved oxygen tension (DOT) in 5 mi-

nute intervals. In these experiments Fab fragment F1 was

expressed in E. coli RV308 in the fed-batch medium. The

polysaccharide substrate in the medium was different

from the previous experiments, and the peptone compo-

nent was replaced by an animal-free peptone. Cultivations

were started with 0.25 g l

-1

glucose and varying concentra-

tions of biocatalyst at pH 7.3. Cultures were induced with

0.2 mM IPTG at 18 h, and at the same time half of the

cultures received Booster and more biocatalyst (3 U l

-1

Cultivations were continued for further 24 h after

induction.

Monitoring of culture growth and pH

Culture growth was monitored by offline cell density

measurements at selected time points. Cell density was

determined by measuring optical density at 600 nm

(OD

600

). OD

600

of 1 corresponds to a dry cell weight of

0.27 g l

-1

. Culture pH level was monitored by offline

measurements of 150 μl broth samples by IQ2400 pH

probe (IQ Scientific).

Determination of Fab expression level

To quantitate the Fab yields, 100 μlbrothsamples

were collected and centrifuged at 13,300 × g and 4°C

for 4 min. The supernatant was collected into a sep-

arate vial, and the cell pellets and supernatants were

both stored at −20°C. For cell disruption the pellets were

thawed, suspended in 100 μl of BugBuster (Novagen) and

lysed by addition of 2 μl Lysonase Bioprocessing Reagent

(Novagen). Cell lysates and broth supernatants were

prepared for analysis by centrifugation at 13,300 × g

and 4°C for 4 min to remove cell debris and other

insolubles.

The quantity of functional Fab in the cell lysates and

broth supernatants was determined by indirect ELISA.

Immuno™96-well MaxiSorp™plates (Nunc) were coated

with 0.1 ml of 1 μgml

-1

thioredoxin-NTproBNP fusion

antigen at 4°C overnight. The wells were washed three

times with PBS + 0.05% Tween-20 (PBST), blocked for

20 min with 1% bovine serum albumin and 0.2% gelatine

in PBST buffer (BSA-gelatine-PBST), and washed again

three times. 0.1 ml of 1:1000 sample dilutions in BSA-

gelatine-PBST were added to the wells and left to bind

for 1 h at room temperature, followed by eight wash

cycles with PBST. Goat anti-mouse IgG (Fab specific) –

alkaline phosphatase (Sigma Aldrich) was diluted 1:5000

in BSA-gelatine-PBST and applied as the secondary anti-

body. The secondary antibody was incubated in the wells

for 30–50 min, followed by seven wash cycles. To detect

alkaline phosphatase activity, 0.1 ml of p-nitrophenyl

phosphate solution (prepared from SIGMAFAST™tab-

lets, Sigma Aldrich) was added to the wells, and the

absorbance at 405 nm was recorded with Thermo

MultiSkan plate reader after 5 to 45 min depending on

the signal strength. To convert the A

405

signal to Fab

concentration in mg l

-1

, purified Fab standards of known

concentration were added to the plate to create a stand-

ard curve for A

405

against mg l

-1

Fab. For each of the

four Fabs, the standard curve was created with a purified

solution of exactly the same Fab as the binding affinities

to the antigen varied widely between the different Fabs.

The periplasmic Fab fraction was analysed from whole

cell lysate with the assumption that all detected Fab

activity originated from the periplasmic space. It is as-

sumed that only correctly folded and biologically active

Fab fragments bound to the antigen and were quantified,

and folding of the Fab to its functional form within cyto-

plasm is generally a very limited occurrence due to the

unfavorable redox conditions. Cytoplasmic assembly to

functional conformation would be virtually impossible

also due to the signal peptide that is only cleaved during

translocation to the periplasm. On these grounds all Fab

activity in the lysate can be accounted to periplasmic

Fab. This was also experimentally verified with a limited

number of cell pellet samples by comparing the Fab

activity in whole cell lysate and periplasmic extract gener-

ated via lysozyme treatment in cold sucrose solution

(30 mM Tris–HCl, 1 mM EDTA, 40% sucrose, pH 8.0;

Neu and Heppel [44]). Two hours incubation in the

lysozyme-sucrose solution at +4°C was sufficient to release

periplasmic proteins without lysing the cells. Analysis by

ELISA confirmed equal yields of functional Fab in the

lysate and the periplasmic extract (data not shown).

Estimation of cell lysis

To estimate the degree of cell lysis in shake flask

cultures, the amounts of total protein in cell pellet

Ukkonen et al. Microbial Cell Factories 2013, 12:73 Page 12 of 14

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and culture supernatant were determined by standard

Bradford microplate assay. Dilutions of bovine serum

albumin were used to create a linear standard curve. 1:100

dilution of samples was sufficient to measure A

595

in the

linear range, and at this dilution the background absorb-

ance by medium components in supernatant samples was

negligible.

Additional files

Additional file 1: Standard deviations of ELISA analysis and

percentages of extracellular Fab F1 in the cultivations with online

DOT monitoring.

Additional file 2: Standard deviations of ELISA analysis,

percentages of extracellular Fab and estimates for cell lysis in shake

flask cultivations.

Additional file 3: Medium pH in shake flask cultivations.

Additional file 4: Standard deviations of ELISA analysis and

percentages of extracellular Fab F1 in small-scale cultivations with

varying culture volumes.

Abbreviations

24dwp: 24 deep well plate; DOT: Dissolved oxygen tension; EB: EnBase;

IPTG: Isopropyl β-D-1-thiogalactopyranoside; k

a: Volumetric oxygen transfer

coefficient; NTproBNP: N-terminal prohormone of brain natriuretic peptide;

OUR: Oxygen uptake rate; PBS: Phosphate buffered saline; SB: Super Broth;

UYF: Ultra Yield Flask; ZYM: ZYM-5052 autoinduction medium.

Competing interests

PN is a co-founder and minor shareholder of BioSilta Oy. PN and AV are

inventors in patent applications EP2226380 A1 and EP2356212 A2. KU and JV

declare no competing interests.

Authors’contributions

KU designed and carried out all cultivations and analyses and wrote the

manuscript. JV constructed the expression vectors, participated in the design

of the study and revised the manuscript. AV participated in the design of the

study and revised the manuscript. PN supervised the study, participated in its

design and data analysis and revised the manuscript. All authors read and

accepted the final manuscript.

Acknowledgements

This work has been supported by a fund from the Finnish Funding Agency

for Technology and Innovation (Tekes) #1695/31/2010 to BioSilta Oy. The

funding source had no role in study design, collection, analysis or

interpretation of data, writing of the manuscript or decision to submit the

manuscript for publication.

We thank Dr Andreas Knepper and Florian Glauche for assistance with the

online oxygen measurements. We thank Professor Heikki Ojamo for critical

reading of the manuscript.

Author details

Department of Process and Environmental Engineering, Bioprocess

Engineering Laboratory, University of Oulu, Oulu, Finland.

BioSilta Oy, Oulu,

Finland.

Department of Physiology, Institute of Biomedicine, University of

Oulu, Oulu, Finland.

Department of Biotechnology, Laboratory of Bioprocess

Engineering, Technische Universität Berlin, Berlin, Germany.

Received: 3 May 2013 Accepted: 16 July 2013

Published: 29 July 2013

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doi:10.1186/1475-2859-12-73

Cite this article as: Ukkonen et al.:Effect of culture medium, host strain

and oxygen transfer on recombinant Fab antibody fragment yield and

leakage to medium in shaken E. coli cultures. Microbial Cell Factories

2013 12:73.

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