This invention relates to the control of expression of a recombinant protein, and more specifically, to a method for screening initial codons providing a desired expression level of the recombinant protein, and methods for controlling the expression and for preparation thereof using the same.

The discipline of proteomics has been mainly considered as an approach allowing for either protein identification from complex mixtures or the characterization of changes at the level of their expresssion/post translational modification(PTM). However, this approach, named forward proteomics, is not sufficient for obtaining an integrated understanding of the coordinated functions of the biological components. Instead, functional characterization of the identified proteins is required to complement the data obtained through the forward proteomic studies, which necessarily involves the expression and analysis of the cloned genes using recombinant-based methodologies. In these functional approaches named reverse proteomics, technology that allows the precise control of recombinant protein expression levels will provide substantial benefits. While it is often desirable to achieve the maximum expression of target proteins, in certain cases, accumulation of recombinant proteins needs to be controlled to prevent potential toxicity or avoid inefficient folding of highly expressed proteins.

Although protein synthesis is a complicated process involving many stages, it is generally accepted that translation of mRNA is mainly controlled at the initiation and/or early elongation phases. In addition to the well-characterized Shine-Dalgarno sequence, statistical studies have identified characteristic distributions of nucleotides around initiation and termination codons. In particular, the sequences of the first few nucleotides in close proximity to the start codon, commonly referred to as the downstream boxes (DB), have been shown to have a significant effect on mRNA translation efficiency. For example, it was demonstrated that the expression level of a lacZ reporter gene could be varied as much as 20-fold depending upon the codon at +2 position (Stenstrom, C. M. et al., Gene 2002, 288(1-2), 1-8). In contrast, it was also shown that the presence of NGG codons (CGG, AGG, GGG, and UGG) in the early region of ORFs significantly repressed the translation of mRNA.

From these results, it is speculated that it might be possible to tune the expression level of given genes by changing their initial codons. While the most straightforward route to do this would be by scanning each position of the 61 codons, the immense time-and labor-requirements make this approach unrealistic, particularly with the conventional in vivo expression technology. For complete coverage of all the possible variant, 3721 (61x61) variants genes would have to be constructed and their subsequent isolation and analysis of the expressed proteins from the same number of cell cultures would also be a daunting task.

The present inventors speculated that it might be possible to tune the expression level of given genes through proper combinations of 'stimulatory' and 'repressive' codons in the initial region of the ORFs. As a result, the inventors have invented a method for obtaining a gene providing a desired level of expression, comprising the steps of preparing randomized initial nucleotide sequences; isolating, expressing and analyzing individual genes with the randomized sequences with both in vivo and in vitro methods; and selecting a codon combination providing the desired expression level and completed the present invention.

Accordingly, an object of the present invention is to provide a method for screening initial codons providing a desired expression level of a protein.

Another object of the present invention is to provide a method for controlling the expression of a recombinant protein using the initial codons screened according to the above method.

A still another object of the present invention is to provide a method for preparing a recombinant protein using the initial codon screened according to the above method.

One aspect of the present invention relates to a method for screening an initial codon providing a desired expression level of a protein, which comprises:

1) randomly changing a gene encoding the protein in one or more positions of codon +2 to codon +13;

2) introducing the randomly changed genes obtained in step 1) into cells, respectively, to isolate them into individual ones, and amplifying them with colony polymerase chain reaction (PCR);

3) performing cell-free protein synthesis from the genes obtained in step 2); and

4) measuring expression levels of the proteins obtained in step 3), and selecting the codon providing a desired expression level of the protein.

Another aspect of the present invention relates to a method for controlling expression of a recombinant protein, which comprises expressing the protein from a gene with the initial codon screened by the above method.

A still another aspect of the present invention relates to a method for preparing a recombinant protein, which comprises expressing the protein from the gene with the initial codon screened by the above method.

The above and other aspects, features and advantages of the disclosed exemplary embodiments will be more apparent from the following detailed description taken in conjunction with the accompanying drawings in which:

Fig. 1 schematically shows an embodiment of the method according to the present invention;

Fig. 2 is a set of graphs showing the results of cell-free protein synthesis of colony polymerase chain reaction (PCR) products;

Fig. 3 is a set of photographs showing the results of electrophoresis of cell-free synthesized protein;

Fig. 4 is a set of photographs showing the results of analysis of steady-state mRNA level;

Fig. 5 is a set of graphs showing predicted mRNA secondary structures;

Fig. 6 is a graph showing the results of expression of EGFP variants; and

Fig. 7 is a set of photographs showing the results of in vivo expression of the selected clones.

Exemplary embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments are shown. This disclosure may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth therein. Rather, these exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of this disclosure to those skilled in the art. In the description, details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the presented embodiments.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Furthermore, the use of the terms a, an, etc. does not denote a limitation of quantity, but rather denotes the presence of at least one of the referenced item. The use of the terms “first”, “second”, and the like does not imply any particular order, but they are included to identify individual elements. Moreover, the use of the terms first, second, etc. does not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another. It will be further understood that the terms “comprises” and/or “comprising”, or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

In the drawings, like reference numerals in the drawings denote like elements. The shape, size and regions, and the like, of the drawing may be exaggerated for clarity.

As used herein, the term “codon +n” indicates a codon located in n ^th position downstream from translation start site. For example, +1, +2, +3, +4, +5 and +6 codons represent first (i.e. start codon, e.g., AT(U)G), second, third, fourth, fifth and sixth codons downstream from translation start site, respectively.

In the present invention, it is attempted to finely modulate the expression level of recombinant proteins by engineering the early codons of the target genes. Specifically, one ore more of +2 to +13 codons, especially +2 and +3 codons of different proteins are changed in a combinatorial manner, and the relationship between the identities of the changed codons and the expression level of the corresponding genes is analyzed. In the present invention, to examine the expression from gene libraries having the randomized initial codons, a strategy of integrating in vivo and in vitro methods is used, which will be specifically explained hereinafter.

Step 1)

One or more of +2 to +13 codons, for example, +2 and +3 codons, of a gene encoding a desired protein is(are) randomly changed. For this, the gene is amplified by polymerase chain reaction (PCR) or other DNA amplification methods with degenerate forward primers having one or more of +2 to +13 codons, for example, +2 and +3 codons, randomized.

Step 2)

The variants obtained in step 1) are introduced into cells to isolate them into individual genes. For instance, the variants are cloned into a vector and transformed into cells, particularly, bacterial cells, more particularly, E. coli cells, Each variant is introduced and transformed into a single cell. Each variant is amplified, for example, by performing colony-PCR using the variant as template.

Step 3)

Each PCR product obtained in step 2) is expressed in a cell-free protein synthesis system. Conventionally, a reaction solution for cell-free protein synthesis comprises cell lysates having cell organelles and factors required for synthesis of the desired protein, amino acid mixture, energy source for protein synthesis, genetic information source and a buffer solution. Examples of cells used to prepare the cell lysates include E. coli , Bacillus subtilis , wheat germ, rice germ, barley germ, CHO cells, hybridoma cells and reticulocytes. Examples of the cell lysates include S30 extract prepared in accordance with the method described in Ahn, J.H. et al., Nucleic Acids Res. 2007, 35(4), e21 and Ahn, J.H. et al., Biochem. Biophy. Res. Commun ., 2005, 338(3), 1346-1352 and S12 extract prepared in accordance with the method disclosed in Korean Patent No. 0733712 (published on June 29, 2007). The amino acid mixture may be a mixture of L-amino acids selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine and glutamine. The energy source for protein synthesis may be one or more selected from the group consisting of ATP, CTP, GTP, TTP and UTP. The genetic information source may be DNA or mRNA encoding the desired protein.

For example, the cell-free protein synthesis may be carried out on a multi-well plate, especially 96-well plate, in an incubator at a temperature ranging from 27 to 37 ℃, especially at 37 ℃, for 1 to 5 hours, especially for 3 hours.

Step 4)

In this step, the expression efficiency is measured for the protein from step 3). In order to measure the expression efficiency of the protein, any conventional method known in the art, for example, radioactivity counting, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the like, may be used.

Fig. 1 schematically shows one embodiment of the method according to the present invention. Briefly, codons +2 and +3 of a gene are randomly changed by performing PCR with synthesized primers having the codons replaced by NNN, and the synthesized mixture of PCR products is cloned into a plasmid, and then, transformed into E. coli DH5α. The transformed cells are cultured in an agar medium, and the genes contained in individual colonies are amplified by colony-PCR to synthesize each PCR product. The PCR products are added to the cell-free synthesis reaction solution to be converted into proteins. The expression of the synthesized protein is quantified by measuring isotope-labeled leucine.

In the present invention, the expression of several hundreds of codon variants was measured for four kinds of proteins. As a result, a continuous distribution of relative expression levels over a 70-fold range was found, enabling the selection of optimal codon combinations for desired expression evels. The results show that change of initial codons, especially +2 and +3 codons, of a gene encoding a specific protein enables the fine-tuning of expression of the protein. Accordingly, the present invention can serve as an effective approach to optimizing protein expression.

The literatures, de Valdivia, E.I.G et al., Nucleic Acids Res . 2004, 32(17), 5198-5205 and de Valdivia, E.I.G et al., FEBS J . 2005, 272(20), 5306-5316, have reported the effects of the initial codons might be related to the drop-off frequency of peptidyl-tRNA from the translating ribosomes.

Considering that the translational ribosome complex is known to be less stable at the beginning of a translated mRNA, codon dependent peptidyl-tRNA drop-off at the very beginning of the coding region in mRNA can lead to a wide range of expression levels. However, the results of this invention show that the initial codons per se did not determine the expression level of the downstream sequence. Instead, it appears that the effect of a given codon is projected in combination with other factors involving the entire sequence of the target genes, which highlights the importance of a high-throughput strategy for screening optimal arrangements of early codons.

The codon dependence of gene expression is not limited to in vitro experiments. The relative expression levels of the +2/+3 variant genes were reproduced when they were expressed in vivo . The codon pairs selected for high expression in vitro remarkably increased protein accumulation in cells, and the medium or low level in vivo expression was also achieved with the in vitro -screened clones. Therefore, the present invention provides an effective means for systematic and precise control of protein production. In addition, the present invention can be applied for the modulation of translation strengths of signal peptide sequences, thereby creating a large repertoire of signal peptides covering a wide range of translation strength, and ultimately, optimizing protein production.

The present invention will be specifically explained with reference to the following examples, which are provided only for the better understanding of the present invention, but should not be construed to limit the scope of the present invention in any manner.

Example 1: Subcloning and colony PCR

In order to control the expressions of four kinds of protein, DsRed2 (AAV97910), EPO (human erythropoietin, CAA26095), UK (serine protease domain of murine urokinase, EDL01484) and EGFP (enhanced green fluorescent protein, AAB02572), codons +2 and +3, immediately followed by the initiation codon ATG, of each gene were changed. Each of the target genes was amplified by PCR with primers having random changes in codons +2 and +3. The primers used were shown in the following Table 1.

(In the above table, the underlined represents a restriction site, and the boldfaced represents an initiation codon.)

The amplified PCR product was cloned into a plasmid (DsRED2, EPO and EGFP into pK7 (see Kim et al., Eur. J. Biochem. , 1996, 239(3) 881-886, and UK into pIVEX2.3d (Roche Applied Science)), respectively, and then, transformed into E. coli DH5α. The transformed cells were cultured in LB-agar plate for one day. Each colony was picked up therefrom and transferred into a PCR reaction solution containing T715up and GTB primers as shown in the following Table 2, and then, colony-PCR was performed. Conditions for colony-PCR were as follows: 30 cycles consisting of 5 minutes at 95 ℃, 30 seconds at 95 ℃, 1 minute at 55 ℃ and 1 minute at 72 ℃, followed by a final extension for 7 minutes.

Example 2: Cell-free protein expression of genes amplified by colony PCR

Each 300 EPO, 222 DsRed2, 300 UK and 120 EGFP PCR product with the +2 and +3 codons of each gene randomly changed was used as an expression template for cell-free protein synthesis. A cell-free protein synthesis system with a volume of 15 μL of comprises as follows: 57 mM HEPES-KOH (pH 8.2), 1.2 mM ATP, 0.85 mM CTP, GTP, UTP, 2 mM DTT, 0.64 mM cAMP, 90 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium acetate, 34μL/mL L-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), 1 mM 20 kinds of amino acids, 0.17 mg/mL E. coli total tRNA mixture (derived from MRE600 strain), 2% PEG(8000), 67 mM creatine phosphate(CP), 5.6 μg/mL creatine kinase, 10M L-[U- ¹⁴ C]leucine(11.3 GBq/mmol, Amersham Biosciences)), 4 μL S30 cell lysate and 1 μL colony PCR products.

S30 lysates were prepared from E. coli BL21-Star ^TM (DE3) (Invitrogen) in accordance with the methods disclosed in the article [Ahn, J.H. et al., Nucleic Acids Res. 2007, 35(4), e21] and [Ahn, J.H. et al., Biochem. Biophy. Res. Commun. , 2005, 338(3), 1346-1352]. After expressing for 3 hours, 15 μL of a reaction sample was taken from the reaction mixture and the expression protein was quantified by measuring radioactivity of the precipitate in trichloroacetic acid (TCA) with a liquid scintillation counter.

The results are given in Fig. 2. As shown in Fig. 2, the expression amounts in all four proteins show broad and continuous distributions. For example, a wild-type EPO gene represents 72 μg/mL of the expression amount while the mutants of +2 and +3 codons show the expression amounts ranging from 8 to 509 μg/mL (see Fig. 2a). The results reconfirm that the initial codon has a significant influence on the expression of the protein. As shown in Figs. 2b to 2d, other three proteins also provided a similar pattern to EPO protein. For the expressions of the colony PCR products, EPO mutant had 11 to 706% of the expression distributions, UK mutant had a 12 to 422% of the expression distribution, DsRed2 mutant had 12 to 834% of the expression distributions and EGFP mutant had 31 to 767% of the expression distributions, as compared to each wild-type, respectively.

Based on the results of [ ¹⁴ C]leucine introduction, 2 μL of the reaction mixtures exhibiting high, moderate or low expression levels were analyzed on 13% tricine-SDS-polyacrylamide gel. The expressed proteins were stained with Coomassie Blue to be visible to the naked eye. The results are given in Fig. 3 (wherein the arrow indicates the expressed protein).

Then, a nucleotide sequence of each clone, five clones exhibiting a high expression level, three clones exhibiting a moderate expression level, and three clones exhibiting a low expression level was analyzed and the results are given in Table 2 (wherein the clones exhibiting a high expression level are indicated as red color, the clones exhibiting a moderate expression level are indicated as blue color, the clones exhibiting a low expression level are indicated as black color, and +2 and +3 codons are indicated as boldface). The expression level is a mean through 3 independent tests and the standard error of the mean in all cases was less than 10%.

As shown in Table 3, it is difficult to identify the common codons increasing or inhibiting the synthesis of the protein in the analysis of the nucleotide sequence; however, it is confirmed that the clones having the higher expression level contain mostly many A and T bases in the initial sequence of gene.

Example 3: mRNA analysis

5 μL was taken from the cell-free protein synthesis solution. The same volume of RNAprotect ^TM bacterial reagent (Qiagen) was added and mixed. RNA was extracted from each sample and isolated with RNeasy mini column and then, the concentration of mRNA was measured on 1.2% formamide agarose gel.

The results are given in Fig. 4. As shown in Fig. 4, the relative concentrations of mRNA are not so different in each gene having different expression level. From the results, it can be demonstrated that the changes of the expression amount according to codon are derived from the efficiency in the protein synthesis process rather than from the synthesis or isolation of mRNA.

Further, the secondary structure of mRNA was analyzed using the Mfold computer program. The results are given in Fig. 5. As can be seen from Fig. 5, there is no relationship between the structural difference of mRNA caused by the change of +2 and +3 codons and the expression amount.

Example 4: Analysis of EGFP activity

In order to analyze a biological activity of EGFP, the amplified EGFP genes (96 EGFP clones) were expressed at 37 ℃ in a 96 well plate on a cell-free system for 3 hours. This process was carried out in the fluorometer and the strengths of the fluorescence of the expressed EGFP were measured in real time (excitation at 488 nm and emission at 555 nm). The strength of the fluorescence of EGFP was observed at 37 ℃ for 1 hour. The fluorescence image photograph of the expressed EGFP was obtained through UV irradiation.

The results are given in Fig. 6. As shown in Fig. 6, it is confirmed that when the PCR products of the mutant of +2 and +3 codons are expressed, the fluorescence increases while the proteins are synthesized, with the lapse of the reaction time. The fluorescence increase rate represented difference in each reaction well and the relative fluorescence strengths were similar to that obtained in the measurement using isotope (see Fig. 2d). The statistic analysis results illustrate that the introduction of [ ¹⁴ C]Leu resulted in a good relationship with EGFP fluorescence (R ² value = 0.971). These results mean that the random mutation at +2 and + 3 codons hardly affects the activity of the synthesized protein.

Example 5: Expression of protein in cell

It was observed whether the effect of the initial codon shown in the cell-free protein synthesis system reappears in a similar tendency in the expression of the protein using the cell. The colony PCR product was subcloned into pET24ma vector (see the article [Yun et al., App. Environ. Microbiol. , 2005, 71(8) 4220-4224]) and transformed to E. coli BL21-Star ^TM (DE3) (Invitrogen) and then, cultured in 5 mL of LB medium. When OD ₆₀₀ reached 0.6, 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) was added thereto and further cultured for 4 hours. A cell culture solution was recovered and expressed proteins were analyzed through 13 % tricine-SDS-PAGE.

The results are given in Fig. 7. As shown in Fig. 7, the results of the expression of the protein using the cell were similar to the effect of the initial codon shown in cell-free protein synthesis system.

Example 6: Confirmation of specificity of initial codon for desired protein

In order to confirm whether +2 and +3 codons showing a higher expression in the special gene also exhibit a similar expression effect in other genes, the expression effects were observed after transfer to other proteins. The results are given in Table 4.

(The number in parenthesis indicates a relative expression effect as compared to a wild-type gene, and the boldface means a sequence of codon exhibiting the highest expression level among four mutant genes.)

As can be seen from Fig. 4, all proteins showed the maximum expression amount in each other different codon combination, respectively. And, there was little increased expression when individual codons were expressed by crossing.

The present invention may be a systematical and exact means for the production of the protein and may assist in better understanding of the complex protein network and in the products of forward proteomics by expressing the protein to the desired level at high speed. Especially, the present invention may be applied to synthesize a signal peptide having various expression strengths by controlling the expression strength of the signal peptide, and consequently may be used to maximize the production of the protein. Further, the present invention may be used in the improvement and advancement of proteins in combination with various synthesis methods by controlling the expression of the protein as well as by mutating the gene at high speed and translating the gene rapidly into a protein.

While the exemplary embodiments have been shown and described, it will be understood by those skilled in the art that various changes in form and details may be made thereto without departing from the spirit and scope of this disclosure as defined by the appended claims.

In addition, many modifications can be made to adapt a particular situation or material to the teachings of this disclosure without departing from the essential scope thereof. Therefore, it is intended that this disclosure not be limited to the particular exemplary embodiments disclosed as the best mode contemplated for carrying out this disclosure, but that this disclosure will include all embodiments falling within the scope of the appended claims.

SEQ ID No. 1 represents the nucleotide sequence of primer F-NNN-DsRED2;

SEQ ID No. 2 represents the nucleotide sequence of primer F-NNN-EPO;

SEQ ID No. 3 represents the nucleotide sequence of primer F-NNN-UK;

SEQ ID No. 4 represents the nucleotide sequence of primer F-NNN-EGFP;

SEQ ID No. 5 represents the nucleotide sequence of primer T715up; and

SEQ ID No. 6 represents the nucleotide sequence of primer GTB.