Báo cáo khoa học: Enhancing the protein production levels in Escherichia coli with a strong promoter potx

Apart from the conditions of temperature and induction, the choice of promoter, bacterial strain and the solubility of the target protein are other parameters that affect total protein p

with a strong promoter

Hanna Tegel, Jenny Ottosson and Sophia Hober

School of Biotechnology, Department of Proteomics, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden

Introduction

Recombinant protein production in bacteria represents

a common strategy for obtaining large amounts of a

protein of interest Although the use of Escherichia coli

has a long tradition in biotechnology, it is still not a

trivial task to determine the optimal production

condi-tions for all proteins A system that is optimal for the

production of one protein might be nonfunctional for

another Apart from the conditions of temperature

and induction, the choice of promoter, bacterial strain

and the solubility of the target protein are other

parameters that affect total protein production, as well

as the amount of soluble protein

Commonly used promoters in E coli include the T7 promoter, which originates from bacteriophage T7 [1] and the E coli lac promoter [2], as well as its modified form lacUV5 [3] The synthetic trc promoter [4], derived from the E coli trp and lacUV5 promoters, is also commonly used The strength of the different promoters is determined by the relative frequency of transcription initiation This is mainly affected by the affinity of the promoter sequence for RNA polymer-ase T7 RNA polymerase is very selective and efficient, resulting both in a high frequency of transcription ini-tiation and effective elongation These features result

Keywords

Escherichia coli; promoter; protein

production; transcription; translation

Correspondence

S Hober, School of Biotechnology, Division

of Proteomics, Royal Institute of

Technology, AlbaNova University Center,

106 91 Stockholm, Sweden

Fax: +46 8 55378481

Tel: +46 8 55378330

E-mail: sophia.hober@biotech.kth.se

(Received 2 July 2010, revised 5 December

2010, accepted 10 December 2010)

doi:10.1111/j.1742-4658.2010.07991.x

In biotechnology, the use of Escherichia coli for recombinant protein pro-duction has a long tradition, although the optimal propro-duction conditions for certain proteins are still not evident The most favorable conditions for protein production vary with the gene product Temperature and induction conditions represent parameters that affect total protein production, as well

as the amount of soluble protein Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target pro-tein In the present study, the effects of three different promoters (T7, trc and lacUV5) on E coli production of target proteins with different charac-teristics are presented The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7 To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount

of soluble protein that is produced In addition, the effects of two different

E coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed It is concluded that the regulation of protein production is

a combination of the transcription and translation efficiencies Other important parameters include the nucleotide-sequence itself and the solubility of the target protein

Abbreviations

ABP, albumin binding protein; eGFP, enhanced green fluorescent protein; His 6 , hexahistidyl tag; PrESTs, protein epitope signature tags;

SD, Shine–Dalgarno.

in an RNA elongation that is approximately five-fold

faster than for E coli RNA polymerase; hence, the T7

promoter is a much stronger promoter than the E coli

promoters [5] The T7 system is also very tightly

regu-lated as a result of the two-step process: the gene

encoding the T7 RNA polymerase that is able to bind

and start transcription from the T7 promoter (the Ø10

promoter from Bacteriophage T7) is positioned in the

E coligenome and governed by the lacUV5 promoter

[1]

Another important criterion when choosing a

suit-able promoter, apart from strength, is the level of

basal transcription A tightly regulated promoter has a

minimal level of basal transcription, which is

particu-larly important if the protein of interest is toxic or

harmful for the host cell [6] A drawback to the trc

promoter is the high basal level of transcription [7] To

further reduce the basal level of the T7 system,

differ-ent approaches could be used For example, a lac

operator could be added downstream of the T7

pro-moter region [8] Another means of regulating the total

mRNA production is via the number of

DNA-copies⁄ plasmids available for transcription To direct

this, different origins of replication [7] are used

The choice of bacterial strain also affects protein

production An E coli strain frequently used for

rou-tine protein production is BL21 [7] To overcome

problems related to recombinant protein production,

this strain has been modified for different purposes

Derivatives of BL21 include strains that decrease the

protease activity and enhance cytoplasmic disulfide

bond formation, as well as strains with a more efficient

protein folding [9] One commonly used BL21 strain is

BL21(DE3) This strain has an insert on the

chromo-some encoding the T7 RNA polymerase controlled by

a lacUV5 promoter This feature allows the use of the

T7 promoter Another problem when producing

human proteins in E coli relates to differences in

codon usage between the two organisms This

differ-ence can lead to translational errors and reduced

production levels of recombinant protein [9] To

overcome the codon bias, genes encoding rare tRNAs

can be co-expressed, as in the case of Rosetta(DE3)

(Novagen, Merck, Darmstadt, Germany)

The solubility of a protein is often of interest in

pro-tein science, especially in structural genomics where

soluble proteins are a requirement for obtaining

infor-mation about the 3D structure [10] Several inherent

parameters affect the solubility of a protein, such as

folding velocity and hydrophobicity When proteins

are produced, the synthesis rate of the protein may

affect the proportion of soluble protein Previously, it

was reported that a decreased protein synthesis rate

(e.g by using a weaker promoter) gives a higher yield

of soluble and correctly folded protein [7]

Great efforts have been made with respect to the development of high throughput methods for the pro-duction and purification of recombinantly produced proteins Different methods for cloning, production and analyses have been developed [11–16] Moreover, purification tags, their positions in relation to the tar-get protein and their effect on productivity and solubil-ity have been evaluated [17] In the present study, the effects of three different promoters (T7, trc and lacUV5) on E coli production of target proteins with different characteristics are presented Protein frag-ments fused to a hexahistidyl tag (His6) and an albu-min binding protein (ABP) were produced, both alone and fused to enhanced green fluorescent protein (eGFP), under the control of the three different pro-moters The total amount of target protein as well as the amount of soluble protein was analyzed, demon-strating the benefits of using a strong promoter such

as T7 To understand the underlying causes, transcrip-tion levels have been correlated with the total amount

of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced In addition, the effects of two differ-ent E coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed

Results

To investigate how different promoters affect protein production and the solubility of the target protein, a set of 16 protein epitope signature tags (PrESTs) was chosen (Table 1 and Doc S1) PrESTs are short regions of human proteins with low similarity to all other human proteins, without transmembrane regions and signal peptides [18] These protein tags are used for immunization aiming to acquire antibodies directed

to the human full-length protein Produced and purified antibodies are used for annotation of the human proteome (relevant data are available at: http:⁄ ⁄ www.proteinatlas.org) The PrESTs were fused with eGFP into vectors with three different promoters; T7, trc and lacUV5 (Doc S2) Upstream of the PrEST, all proteins contained a His6-tag followed by ABP All constructs were transformed into E coli BL21(DE3) and fifteen of the constructs also into

E coli Rosetta(DE3) Protein production in shake flasks was performed to assess the different expression patterns It was not necessary to use BL21(DE3)-based strains when proteins were produced under the control

of the trc and lacUV5 promoters because the main purpose of the strain modifications was to create an

inducible expression of T7 RNA polymerase However,

to minimize the differences in behavior both during

cultivation and in the fluorescence activated cell

sort-ing measurements, the same strain was used for all

promoters In addition to the direct induction of the

trc and lacUV5 promoters, expression of T7 RNA

polymerase is anticipated but, because T7 RNA

poly-merase by itself is not toxic to the E coli cells and

only recognizes the T7 promoter, this should not

inter-fere with the transcription initiated by the trc and

lacUV5 promoters [1]

Analysis of the total amount of produced protein

For analysis of protein production, cells from the

cul-tures were disrupted and separated into a soluble and

an insoluble fraction by centrifugation Both fractions

were analyzed by SDS⁄ PAGE and western blotting

using quantityone software (Bio-Rad Laboratories,

Hercules, CA, USA) (Fig 1A) The amount of target

protein was correlated with the amount of cells loaded

and to protein samples with a known concentration

The relative amount of produced protein, normalized

according to cell density, is presented in Table 1 As expected, the data show that protein production under the control of the T7 promoter gives the largest total amount of target protein, whereas lacUV5 gives the lowest A large difference between different proteins produced under the control of the same promoter could also be detected

To determine whether the transcription rate is only dependent on the three different promoters or whether the transcription rate is also sequence-dependent, real-time PCR was used to compare the number of mRNA molecules before and after induction Even more importantly, the impact of mRNA levels on protein production was investigated Five His6 -ABP-PrEST-eGFP constructs (chosen to represent proteins with different solubilities and production levels) under the control of the three different promoters were produced and samples were taken to determine the fold change

of mRNA caused by the induction Figure 1B shows that the fold change of mRNA after induction is corre-lated with the amount of target protein that is pro-duced Again, all data were normalized according to cell density As seen in Fig 1, the transcription levels

Table 1 Summary of the proteins, their characteristics and production levels in E coli BL21(DE3) Proteins A–P correspond to the PrEST part of the His 6 -ABP-PrEST-eGFP fusion protein For the exact nucleotide and amino acid sequences of each PrEST, see Doc S1 Solubility class is defined as described in the Materials and methods, with group 1 as the most insoluble and group 5 as the most soluble The sym-bols shown are the same as those used in Figs 1 and 3 The amount of produced protein for 17 different fusion proteins under the control

of three different promoters is summarized In addition, the amount of soluble target protein is shown All values for the amount of protein are adjusted to cell density and normalized to the highest production value (total amount for protein F under the control of the T7 promoter) The fraction of soluble protein is shown on the right The average error based on two separately cultured samples was 0.031 (T7), 0.011 (trc) and 0.084 · 10)3(lacUV5) for the total amount of protein; 0.0012 (T7), 0.00049 (trc) and 0.057 · 10)3(lacUV5) for the amount of soluble protein; and 0.0075 (T7), 0.0034 (trc) and 0.027 (lacUV5) for the soluble fraction NA, Not Applicable.

Protein

Accession

number

(Uniprot)

Gene name

Solubility class

Symbol

Total (· 10 3 ) Soluble (· 10 3 ) Soluble fraction (%) Without

eGFP

With

eGFP

are dependent on the promoter used, and the relative

order of these appears as expected, with the lacUV5

promoter giving the lowest change of mRNA level and

the T7 promoter the highest However, the differences

among the constructs including the T7 promoter are

larger than expected both with respect to changes in

mRNA levels and the correlation between the amount

of mRNA and protein With respect to mRNA con-centration, protein L under the control of the T7 pro-moter showed a much higher fold change than the other proteins When repeated, the analyses resulted in diverse data for this protein, although the average fold change for protein L was clearly higher than for the other proteins One consideration worthy of note when studying the result shown in Fig 1B is the high level

of basal transcription (promoter leakage) caused by the trc promoter Because of this leakage, the analyzed differences in mRNA levels most probably are a slight misrepresentation of the total mRNA levels within the cell at harvest

One reason for the spread in the amount of protein that is produced could be the number of rare codons, which might stall the ribosome when translating the mRNA to an amino acid sequence Therefore, we also analysed the codon composition of the different pro-teins (Table 2) Both propro-teins J and O, which have a higher relative amount of produced protein, have a few rare codons, especially rare arginine codons Hence, the translation process in E coli BL21(DE3) is probably faster for these proteins than for proteins containing a higher amount of rare codons

Analysis of the amount of soluble produced protein

Apart from the analyses aiming to determine whether the total amount of produced protein is affected by different promoters, the present study investigated how different promoters affect the amount of soluble tein obtained Therefore, the fraction of soluble pro-tein was analyzed Interestingly, the weakest promoter generates the largest fraction of soluble protein and vice versa and, generally, the fraction of soluble pro-tein is very small when propro-teins are produced under the control of T7 or trc (Table 1) However, three of the proteins (B, D and I) differ from the rest regarding these aspects B and D both show a relatively large fraction of soluble protein when produced under the

Insoluble fraction Soluble fraction

T7 trc lacUV5 T7 trc lacUV5

97.0

66.0

45.0

30.0

20.1

14.4

Target protein

100

120

Relative amount of produced protein

0

20

40

60

80

140

Relative amount of produced protein

0

2

4

6

8

10

12

14

0.002 0.001 0.000 0.003

0.6 0.2

A

B

Fig 1 Analysis of the total target protein production in E coli

BL21(DE3), adjusted to cell density The mean amount of produced

target protein was 6.6 mgÆ100 mL)1 culture for T7;

2.3 mgÆ100 mL)1 culture for trc; and 12 lgÆ100 mL)1 culture for

lacUV5 For an explanation of protein symbols, see Table 1 (A) An

example of a representative SDS ⁄ PAGE for determination of

pro-tein production levels, western blotting (upper) and Coomassie

stain (lower) analysis In each analysis, the insoluble and soluble

fractions of six cell samples were analyzed For western blotting,

the insoluble T7 and trc fractions were diluted 1 : 1000 and the

sol-uble T7 and trc fractions were diluted 1 : 100 As a marker in the

western blotting, a protein of known concentration was used;

100 ng was loaded in the first marker lane and 10 ng in the second

marker lane Low molecular weight markers were used to identify

protein sizes in the gel The target protein is indicated by an arrow.

(B) The correlation between mRNA fold change and amount of

pro-duced protein, normalized to the highest value, for five proteins

under the control of the three promoters The fold change was

cal-culated as the mean of three separate experiments in all cases but

one For protein E under the control of lacUV5, an outlier by a

fac-tor of 7.8 was excluded Light grey, black and grey represent the

T7, trc and lacUV5 promoters, respectively Inset: magnification

showing data points representing the proteins that are produced

the under the control of the lacUV5 promoter.

Table 2 Summary of results from the codon analysis.

Protein

Number

of codons

Number

of rare codons

Number of AGG and AGA codons

control of the stronger promoters When the trc

pro-moter is used, these two proteins show equally large

fractions of soluble protein, whereas D is the only

pro-tein with a large soluble fraction under the control of

T7 On the other hand, protein I appears to be very

insoluble even under the control of lacUV5

Although the fraction of soluble protein is very

interesting, it is still the amount of soluble protein that

is most important Table 1 shows the relative amount

of soluble protein correlated with cell density It is

clear that, even though lacUV5 gives the largest

frac-tion of soluble protein, T7 is the promoter that gives

the largest amount of soluble protein

Impact of the solubility of the protein on the

amount of soluble produced protein

Because one aim of the present study was to assess

information about protein solubility during protein

production, the PrEST proteins used were chosen with

the aim of covering a large span of different protein

solubilities when produced as a fusion of His6

-ABP-PrEST One method that we wanted to use for the

assessment of in vivo solubility was flow cytometric

analysis, which takes advantage of the

solubility-dependent fluorescence of GFP Because eGFP was

fused to the C-terminus of the protein, it was of great

importance to determine whether eGFP affects the

sol-ubility of the different target proteins The fusion

pro-teins were therefore produced with and without eGFP,

followed by immobilized metal ion affinity

chromatog-raphy purification to determine the solubility by using

an in vitro solubility test [19] All proteins were graded

from 1 to 5 Class 1 constitutes the most insoluble

pro-teins and class 5 represents the most soluble propro-teins

As shown in Table 1, eGFP generally decreases the

solubility of proteins belonging to classes with a high

solubility and increases the solubility of proteins

belonging to classes with a low solubility without

eGFP In other words, eGFP appears to be a burden

for highly soluble proteins, whereas it can increase the

solubility of a poorly soluble protein

The correlation between the amount of soluble

pro-duced protein and in vitro solubility data was assessed

(Fig 2) Data providing information about the amount

of soluble produced protein was obtained from the

SDS⁄ PAGE and western blotting analyses and

com-pared with the data obtained when analyzing the same

protein in vitro Because eGFP does affect the

solubil-ity, the solubility class used in this case is the one with

eGFP As shown in Fig 2, there is a slight positive

correlation between the relative amount of soluble

protein and solubility class in vitro The proteins with

higher protein solubility class are more likely to yield a higher amount of soluble protein Interestingly, this correlation is independent of the choice of promoter

Comparison of protein production in E coli BL21(DE3) versus Rosetta(DE3)

Because the PrEST parts of the fusion proteins are derived from the human genome and there is a codon difference between human and E coli, it is interesting

to determine whether the expression pattern differs when the production is made in E coli Rosetta(DE3),

a strain that, as a result of additional genetic informa-tion, compensates for the tRNAs commonly used by eukaryotes Five of the fusion proteins, under the con-trol of all three different promoters, were therefore transformed into Rosetta(DE3) cells, produced and analyzed The total amount of produced protein was analyzed and compared with the results obtained after production in BL21(DE3) cells As shown in Table 3, the two strains give the same expression pattern when comparing the different promoters with each other However, Rosetta(DE3) generates a larger amount of produced protein irrespective of the promoter In an attempt to explain the increased production when using Rosetta(DE3), the occurrence of rare codons within each PrEST sequence was compared with the amount of produced protein, although no obvious correlation was found (data not shown)

The fraction of soluble protein after production in Rosetta(DE3) was compared with the data obtained with respect to production in BL21(DE3) As shown in Table 3, independent of the strain, lacUV5 gives the largest fraction of soluble protein; however, of even more interest is a comparison of the amount of soluble

Solubility class with eGFP

T7

trc lacUV5

1.2

0.8

0.6

0.4 1.0

0.2

0.0

Fig 2 The correlation between the relative amount of soluble protein in E coli BL21(DE3), normalized to the highest value, and the in vitro solubility class with eGFP.

protein after production in BL21(DE3) and Roset-ta(DE3) From a comparison of the data provided in Table 3, it is obvious that, even in this respect, it is beneficial to use Rosetta(DE3) rather than BL21(DE3)

If the desired goal is the highest possible amount of soluble protein, the strain Rosetta(DE3) is the best choice Possibly more interesting is the changed expression pattern As can be seen from Table 3, the combination of the trc promoter and the Rosetta(DE3) strain gives more soluble protein than T7 and Rosetta(DE3) in three out of five cases

It was previously shown that the levels of soluble protein can be determined, during protein production

in vivo, by using a flow cytometer Proteins are fused to the N-terminus of eGFP and the cells producing these fusion proteins can then be analyzed [20] This method was used to further assess the production in BL21(DE3) and Rosetta(DE3) Thus, after protein pro-duction in the two different strains, the cells were ana-lyzed by using a flow cytometer The behavior in the flow cytometer correlates well with the amount of solu-ble protein (Fig 3) Interestingly, the strain appears to affect the signal achieved because two populations are formed Figure 3 clearly shows that the whole cell fluorescence after production in BL21(DE3) is higher than in Rosetta(DE3), although the amount of soluble protein is similar By using this alternative method, the results shown in Table 3 could be confirmed Roset-ta(DE3) is favorable if soluble protein is desired

Soluble fraction

Soluble fraction

Soluble fraction

Relative amount of soluble protein

0.7

0.6

0.5 0.4 0.3

0.2 0.1

0.0 0.6

0.4

0.2

0.0

0.8 1.0 1.2

Fig 3 Solubility analysis of eGFP fusion proteins The correlation between whole cell fluorescence and amount of soluble protein, normalized to the highest value, for 30 cell samples Five proteins were produced under the control of three different promoters in two bacterial strains: E coli BL21(DE3) and Rosetta(DE3) The filled data points represent the proteins that are produced in BL21(DE3) and the unfilled data points represent the proteins that are pro-duced in Rosetta(DE3) The data are based on measurements per-formed with two separately cultured samples The average error in the fluorescence activated cell sorting analysis was 13% Two pop-ulations of different fluorescence, depending on the choice of

E coli strain, are indicated by trend lines For an explanation of the different symbols used, see Table 1.

In the flow cytometric analysis, the production in

BL21(DE3) of some additional samples was analyzed

Except for three samples, they all showed the same

correlation as the BL21(DE3) population in Fig 3

The outliers all had a large amount of soluble protein

without showing any whole cell fluorescence To

deter-mine whether this was caused by an inactive but

solu-ble eGFP, the eGFP activity of purified protein from

the soluble fraction was studied The three outliers did

not show any eGFP activity, as was the case for the

positive control (data not shown) An additional

evalu-ation of the correlevalu-ation between the amount of soluble

and insoluble protein achieved was performed for this

data set A constant ratio was seen between the two

protein fractions for almost all proteins when using the

T7 and trc promoters, regardless of the strain used

(data not shown) Interestingly, there are two proteins

(A and E) that show a larger fraction of soluble

pro-tein than the other propro-teins when produced in

Roset-ta(DE3) under the control of the trc promoter For

proteins produced under the control of lacUV5, the

amount of insoluble protein is generally low and an

increased protein production gives mostly soluble

pro-tein In other words, lacUV5 has a larger fraction of

soluble protein, although, as an effect of the low total

production, the amount of soluble protein is much

lower than for T7 and trc

Discussion

To further understand the effect of the promoter on

the acquired protein, 17 different proteins have been

produced under the control of three different

promot-ers Because the final amount of protein achieved also

is dependent on other important features, such as

mRNA stability, transcription and translation

efficien-cies, and protein stability, a comparison of the total

amount of protein as well as the fraction of soluble

protein achieved with different promoters was

ana-lyzed for 17 different proteins with different

character-istics, pI and solubility As expected, the data show

that a strong promoter is a benefit when a large

amount of protein is desired (Table 1) Noteworthy,

when comparing the mRNA level with the amount of

protein achieved, a high correlation between these

parameters could be seen (Fig 1B) Hence, the weak

lacUV5 promoter shows a low fold change as well as

low protein production compared to the stronger

pro-moters, trc and T7, which both show higher values

Interestingly, there are some proteins that do not

fol-low the expected pattern A fol-lower protein production

than expected could be an effect of poor mRNA

stability or proteolysis within the cell However, to

minimize proteolytic effects, we limited the induction time to 3 h [20] Accordingly, the bacteria should not experience any limitations with respect to oxygen sup-ply or nutrition Both proteins J and O show a larger amount of produced protein under the control of the T7 promoter than expected This behavior could be explained by these mRNA molecules being more effec-tively translated as a result of having few rare codons, especially a low number of the rare arginine codons (Table 2) One way to compensate for differences in codon usage is by co-expression of genes encoding rare tRNAs; for example, by using the E coli strain Rosetta(DE3) When comparing the protein produc-tion of five different proteins in E coli BL21(DE3) with the production in Rosetta(DE3), the Roset-ta(DE3) strain generated a higher amount of protein for all three promoters (Table 3) However, as in a previous study carried out by Tegel et al [21], the benefit of using Rosetta(DE3) could not be explained solely by the number of rare codons within the trans-lated genes (data not shown) Also, the efficiency of different tRNA synthetases and the 3D structure of the translated mRNA may effect the translation effi-cacy These conclusions were also drawn by Welch

et al [22] Surprisingly, in three of five cases, the com-bination of Rosetta(DE3) and the trc promoter gives more soluble protein than does Rosetta(DE3) and the T7 promoter (Table 3) However, in the other two cases, the T7 promoter gave substantially larger amounts of target protein

With respect to translation, one parameter that is even more important for overall translation efficiency than codon usage is the efficiency of translation initia-tion This step is mainly influenced by features related

to the Shine–Dalgarno (SD) sequence, such as the sequence itself, the length of the sequence and the dis-tance between the SD sequence and the initiation codon Within the SD sequence used in the expression vectors in the present study, some differences could be observed The most obvious differences are the sequence itself and the sequence length The SD sequence in the T7 vector, AAGGAG, is longer than the one used in the lacUV5 and trc vectors, AGGA (Doc S2) A study by Ringquist et al [23] concluded that the SD sequence UAAGGAGG initiates transla-tion approximately four-fold more efficiently than AAGGA Comparing these sequences with the SD sequences used in the present study, the translation efficiency will most likely be higher for mRNA tran-scribed from the T7 vector In other words, the same number of mRNA molecules could generate different amounts of protein depending on the SD However, in the present study, the correlation between the fold

change in mRNA levels and the amount of protein

indicates that the differences between the translation

efficiency for different SD sequences are rather small

Moreover, if the leakage of the trc promoter is taken

into account, the final concentration of mRNA for this

vector is even higher, which indicates that the

transla-tion efficiency of the SD sequence included in the T7

promoter is no higher than for the other vectors One

explanation for this could be that the T7-driven

tran-scription is uncoupled from translation and proceeds

several times faster than the ribosomes are able to

fol-low Hence, the transcribed mRNA is not as efficiently

used for translation as those that exhibit a coupled

transcription⁄ translation activity [24]

Depending on the final application of the produced

protein, the need for soluble protein differs As shown

in Table 1, the largest fraction of soluble protein is

generated by lacUV5, which is the weakest promoter

However, when it comes to the amount of soluble

pro-tein, the two stronger promoters are beneficial as a

result of higher total production The T7 promoter

should therefore also be used when large amounts of

soluble protein are desired The larger fraction of

solu-ble protein generated by lacUV5 is explained by the

weaker promoter giving a lower protein synthesis rate

as a result of less mRNA, and thereby each protein

has more time to fold correctly and form a soluble

protein before forming an insoluble protein precipitate

by colliding with other recently translated proteins

Even though the majority of all proteins had a large

fraction of soluble protein under the control of

lacUV5, protein I was shown to be very insoluble

regardless of the promoter By contrast, proteins B

and D appeared to be more soluble than the other

proteins when produced under the control of trc and

T7 One explanation for this might involve differences

in folding rate or the structural features of the

trans-lated protein The differences in the fractions of

solu-ble protein achieved for the different proteins could, in

most cases, also be correlated with the solubility of the

protein itself

Hedhammar et al [20] has previously shown that

the levels of soluble protein within the cell could be

determined using a flow cytometer In the present

study, we show that this correlation is highly

depen-dent on the strain used for protein production (Fig 3)

In addition, there might be soluble proteins with

inac-tive eGFP resulting in misleading results Moreover, it

has also been shown that GFP captured in inclusion

bodies also could contribute to the measured

fluores-cence [25] However, the high correlation between

fluo-rescence and the amount of soluble protein shown in

the present study indicates that the main part of the

measured fluorescence originates from correctly folded and soluble protein

Finally, we conclude that the regulation of protein production is a combination of the transcription and translation efficiencies Other important parameters include the gene itself and the solubility of the protein

A general recommendation, if a large amount of pro-tein is needed, is to use the T7 promoter in combina-tion with the Rosetta(DE3) strain If the amount of soluble protein is important, protein production should

be performed in Rosetta(DE3) cells under the control

of the T7 or trc promoter

Materials and methods Materials and strains

All recombinant work was performed in E coli strain RR1DM15 [26], essentially as described by Sambrook et al [27] Oligonucleotides for cloning of the different constructs were purchased from MWG-biotech AG (Edersberg, Ger-many), whereas the oligonucleotides for real-time PCR were purchased from Thermo Electron GmbH (Ulm, Germany) Restriction enzymes were manufactured by New England Biolabs (Ipswich, MA, USA) and ligase by Fermentas Life Sciences (Vilnius, Lithuania) All enzymes were used in accor-dance with the manufacturers’ instructions To sequence ver-ify the constructs, an ABI Prism 3700 DNA sequencer (Applied Biosystems, Foster City, CA, USA) was used Plas-mids were purified using Qiaprep Spin Miniprep kit (Qiagen GmbH, Hilden, Germany) Production of the fusion proteins was performed in E coli strain BL21(DE3) and E coli strain Rosetta(DE3) (co-expression of tRNA genes for AGG, AGA, GGA, AUA, CUA and CCC) (Novagen)

Cloning

DNA sequences coding for the promoters lacUV5 and trc were amplified by PCR from vectors including the relevant genes By using primers TEHA1: ACACAGATCTCTGCA-GGGCACCCCAGGCTTTACA and TEHA2: ACACCC-ATGGAGCTTTCCTGTGTGAAATTGT, lacUV5 was amplified TEHA3: ACACAGATCTCTGCAGTGAAATG-AGCTGTTGACAATTA and TEHA4: ACACCCATGGT-CTGTTTCCTGTG were used for trc amplification The exact nucleotide sequence of each promoter region is pro-vided in Doc S1 A common handle sequence introduced the restriction sites for BglII and PstI upstream and NcoI downstream of the promoters The resulting PCR frag-ments were digested with BglII and NcoI and ligated into pAff8eGFP (with a pBR322-ori and encoding kanamycin resistance) [20], cut with the same enzymes and thereby replacing the sequence encoding the T7 promoter, using solid-phase cloning [18] The resulting vectors were

sequence verified and named pAff8eGFPLacUV5 and

pAff8eGFPTrc, respectively

The gene for the T7 promoter was amplified from the

vector pAff8eGFP using TEHA7:

ACACCTGCAGCGAT-CCCGCGAAATTAATAC and TEHA8:

ACACCCATGG-TATATCTCCTTCT, introducing restriction sites for PstI

upstream and NcoI downstream of the promoter The PCR

fragment and pAff8eGFPTrc were digested with PstI and

NcoI before the PCR fragment was ligated into the cut

vec-tor using solid-phase cloning, replacing the trc with the T7

promoter The resulting vector was sequence verified and

named pAff8eGFPT7

Sixteen different PrESTs (Table 1) were PCR-amplified

from the pAff8cPrEST [18] plasmids using primers

intro-ducing an upstream NotI site and a downstream AscI site,

although without introducing a downstream stop codon

The PCR products were digested with NotI and AscI and

ligated into pAff8eGFPT7, pAff8eGFPTrc and

pAff8eG-FPLacUV5 using solid-phase cloning, resulting in plasmids

encoding His6-ABP-PrEST-eGFP under the control of three

different promoters All constructs were transformed into

E coli strain BL21(DE3) and some of them also into

E colistrain Rosetta(DE3)

Protein expression

One milliliter of overnight culture in tryptic soy broth

(Merck KGaA, Darmstadt, Germany), 30 gÆL)1,

supple-mented with 5 gÆL)1 yeast extract (Merck KGaA,

Darms-tadt, Germany) and 50 lgÆmL)1kanamycin (Sigma-Aldrich,

Munich, Germany) was used to inoculate 100 mL of

identi-cal media in 1 L Erlenmeyer flasks When using the E coli

Rosetta(DE3) strain for protein production, 20 lgÆmL)1

chl-oramphenicol was also added to the culture media The

cul-tures were incubated on shakers (150 r.p.m.) at 37C until

OD600of 0.5–0.8 was reached Protein production was then

induced by addition of isopropyl thio-b-d-galactoside

(App-ollo Scientific Ltd, Stockport, UK) to a final concentration

of 1.0 mm Incubation continued at 30C for 3 h The cells

were harvested by centrifugation (2400 g for 8 min at 4C)

and the pellet was re-suspended in 30 mL of 1· PBS (20 mm

NaH2PO4, 80 mm Na2HPO4, 150 mm NaCl) At harvest,

the cell density varied between 3.9 (for T7) and 5.2 (for trc),

with a mean of 4.5

Analysis of the total and soluble protein

production

SDS⁄ PAGE and western blotting

To be able to fractionate the soluble and insoluble proteins,

the cells were disrupted by sonication at 60% duty cycle for

3 min with 1.0 s pulses (Vibra cell; Sonics and Materials,

Inc., Danbury, CT, USA) The sonication level was

evalu-ated using viable count One milliliter of the sonicevalu-ated cells

was centrifuged for 10 min at 9500 g in a microcentrifuge

to separate the soluble from the insoluble proteins The pel-lets were then washed twice with 200 lL of 1· NaCl ⁄ Piand the washing solution was added to the soluble fraction To concentrate all soluble fractions, lyophilization (Automatic Environmental SpeedVac system AES2010; ThermoSavant, Holbrook, NY, USA) was used Both soluble and insoluble fractions were then diluted to the same volume and all frac-tions were analyzed on Criterion Precast SDS⁄ PAGE 10– 20% gradient gels (Bio-Rad Laboratories) and stained with GelCode Blue Stain Reagent (Thermo Scientific, Rockford,

IL, USA) in accordance with the manufacturers’ instruc-tions The gels were destained with distilled water before scanning at 400 d.p.i

To be able to detect low producing proteins, all fractions were also analyzed on western blots After SDS⁄ PAGE separation, the proteins were electroblotted onto a polyvinylidene fluoride membrane (Criterion Gel Blotting Sandwiches; Bio-Rad Laboratories) in accordance with the manufacturer’s instructions The blotted proteins were detected using a Ni-NTA horseradish peroxidase conjugate (Qiagen GmbH) in combination with SuperSignal West Dura extended duration substrate (Thermo Scientific) in a ChemiDoc CCD camera (Bio-Rad Laboratories), all in accordance with the respective manufacturers’ instructions All gels and western blots were evaluated using quantityone 4.6.3 software (BioRad Laboratories) The bands of the recombinant proteins, both soluble and insolu-ble, were normalized against some of the soluble E coli house-keeping proteins that are produced equally in all cells

Real-time RT-PCR

Samples were taken from the cultures before induction and

at harvest The total RNA from the bacteria was purified using RNeasy Protect Bacteria Mini Kit (Qiagen) Two sep-arate cDNA synthesis reactions were performed for each total RNA: synthesis of the reference gene (ribosomal protein rpmE) and the target gene (eGFP) using reverse-specific primers, rpmE_R: GGGATGTTGAAACGCTT GTTG and GFP6_R: CGGTCACGAACTCCAGCAG, respectively The input of total RNA was 2 lg A mixture containing total RNA, dNTPs (Invitrogen, Carlsbad, CA, USA) and 5 pmol of each reverse primer was denatured at

70C for 10 min and then cooled on ice for 2 min Subse-quently, 200 units of SuperScript III reverse transcriptase (Invitrogen) were added and cDNA synthesis was per-formed at 46C for 1 h The enzyme was inactivated at

85C for 5 min The total volume of the cDNA synthesis reaction was 20 lL and contained 0.25 lm specific primer, 0.5 mm dNTP, 5 mm dithiothreitol (Invitrogen) and 1· First-Strand Synthesis Buffer [50 mm Tris-HCl (pH 8.3),

75 mm KCl, 3 mm MgCl2; Invitrogen]

Real-time PCR was performed with an iCycler iQ 3.0 (Bio-Rad Laboratories) in 25 lL reactions containing

12.5 lL of iQ SYBR Green Supermix (Bio-Rad

Laborato-ries), 5 lL of cDNA template and 5 pmol of reverse

(rpmE_R, GFP6_R) and forward (rpmE_F:

AAGTGCCA-CCCGTTCTTCAC, GFP6_F:

GACAACCACTACCTGA-GCAC) specific primers PCR amplification was carried out

at 95C for 30 s followed by 35 annealing and extension

cycles (94C for 20 s, 62 C for 30 s and 72 C for 1 min)

After the amplification, a melt curve analysis was

per-formed by ramping the temperature from 60C to 100 C

The obtained CT values of the analysis were then

deter-mined using icycler Software (Optical System Software,

version 3.0a) The CTvalues were converted into the fold

change data using the 2)DDCTmethod [28]

In vitro solubility assay

His6-ABP-PrEST proteins, with and without eGFP, were

purified by immobilized metal ion affinity chromatography

[29] using a fully automated purification set-up [30] The

in vitrosolubility of each recombinant protein was assessed

using a method developed by Stenvall et al [19] The

con-centration of all purified proteins was adjusted to

0.8 mgÆmL)1 in 1 m urea All samples were then diluted

five-fold in 1· NaCl ⁄ Pi resulting in a final urea

concentra-tion of 0.2 m Immediately after diluconcentra-tion, the initial protein

concentration was determined using the bicinchoninic acid

kit (Thermo Scientific) Thereafter, the samples were

incu-bated at 30C for 20 h After incubation, the precipitated

proteins were separated from the soluble proteins by

centri-fugation at 2800 g followed by a second concentration

determination of the soluble fraction The difference

between the two measurements corresponds to the amount

of precipitated protein The proteins were classified from 1

to 5 depending on the degree of precipitation, where grade

1 was the least soluble (80–100% precipitation), followed

by grade 2 (60–80% precipitation), grade 3 (40–60%

pre-cipitation) and grade 4 (60–80% prepre-cipitation), with grade

5 being the most soluble (0–20% precipitation) [19]

Flow cytometric analysis

The flow cytometric analysis was performed on a FACS

Vantage SE stream-in-air flow cytometry instrument (BD

Biosciences, San Jose, CA, USA) To align the laser flow

cytometry alignment beads for 488 nm (Molecular Probes,

Leiden, The Netherlands) were used Samples, containing

whole cells diluted 1 : 100 in 1· NaCl ⁄ Pi, were illuminated

with an air-cooled argon ion laser (488 nm) The

fluores-cence from 10 000 cells was detected at a rate of

approxi-mately 500–750 eventsÆs)1 via a 530 ± 15 nm (green) band

pass filter The analytical flow cytometric histograms were

recorded using standard procedures cellquestpro

software (BD Biosciences) was used to analyze the flow

cytometric data E coli BL21(DE3) cells producing

His6-ABP-eGFP and His6-ABP-SOD1 under the control of

the T7 promoter were used as positive and negative controls, respectively, in each analysis The relative fluores-cence for each construct was normalized with the two controls [20]

Acknowledgements The authors would like to thank Dr C Agaton, Dr

M Hedhammar, Mrs C Asplund and Dr J Steen for fruitful discussions and technical assistance The authors would also like to thank the referees for their construc-tive comments that helped to improve the manuscript This work was financially supported by grants from the Knut and Alice Wallenberg Foundation

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