SE544059C2 - Synthetically evolved dna constructs for regulating signal peptide performance as well as vectors and host cells thereof - Google Patents

Abstract

The present invention provides a simple and inexpensive system for regulating signal peptide performance by using a synthetically evolved nucleotide sequence. The invention further relates to an expression vector comprising the nucleotide sequence. Additionally, the present invention relates to host cell comprising the expression vector. Furthermore, the present invention relates to a recombinant protein expressed by the host cell as well as a method for expressing the recombinant protein.

Description

SYNTHETICALLY EVOLVED DNA CONSTRUCTS FOR REGULATING SIGNAL PEPTIDE PERFORMANCE AS WELL AS VECTORS, HOST CELLS AND RECOMBINANT PROTEINS THEREOF TECHNICAL FIELD The present invention relates to the general field of regulating signal peptide performance. More specifically, the present invention relates to regulating recombinant protein expression via controlling signal peptide performance.

BACKGROUND Bacterial cell factories are widely used in the biotech and pharmaceutical industries for the production of high-value recombinant proteins. Classic examples include industrial enzymes, hormones and antibody fragments, which generate billions of dollars in revenue annually [1,2]. These recombinant proteins are typically engineered with an N-terminal signal peptide so that they will be secreted out of the bacterial cytoplasm [3]. For industrial enzymes, which are usually produced in gram-positive bacteria such as Bacillus subtilis, secretion from the cytoplasm to the culture supernatant simplifies purification and downstream processing. For hormones and antibody fragments, which are usually produced in gram-negative bacteria like Escherichia coli, secretion from the cytoplasm to the oxidizing environment of the periplasm is necessary for the formation of disulfide bonds that are essential for protein folding and activity [4,5].

Secretion out of the bacterial cytoplasm is usually mediated by the general secretion pore (Sec) [6,7], Sec is a major hub for protein trafficking as it inserts proteins into the cytoplasmic membrane, and secretes proteins to the envelope and beyond. Secreted proteins are typically targeted to Sec by an N-terminal signal peptide. Signal peptides vary in length and amino acid sequence, but have a distinctive tripartite structure that includes a positively-charged N-terminal region, a hydrophobic core, and a polar C-terminal cleavage site that typically contains the signal peptidase recognition site (Ala-X-Ala) [8,9]. They also have a distinctive codon usage, which includes a biased use of the AAA (Lys) codon at the second position, and a high frequency of non-optimal codons [10-14], It has been suggested that the signal peptide slows folding of the protein in the cytoplasm and targets it to Sec in a predominantly unfolded confirmation [15]. Upon arrival at Sec the signal peptide also promotes binding to the SecA chaperone, thereby allosterically activating Sec for protein secretion [16]. Given these multiple roles it is likely that signal peptides have co-evolved with the protein that they translocate, as well as with the secretion machinery.

Signal peptides have unpredictable effects on the production yields of recombinant proteins. For example, a signal peptide that supports a high-level of protein synthesis and secretion for one recombinant protein often supports a low-level of protein synthesis and secretion for another. Herein we refer to this phenomenon as signal peptide performance (i.e. signal peptide strength). Since it is not possible to predict how well a signal peptide will perform with a given recombinant protein, it is common practice to screen large signal peptide-libraries for one that supports a high-level of protein synthesis and secretion [3]. This approach is both time-consuming and expensive. Hence, there is a need for a molecular understanding of signal peptide performance since as it could lead to new methods for (1) identifying suitable signal peptides, and (2) rationally engineering signal peptides that increase production yields in bacterial cell factories.

Translation initiation is a rate-limiting step of protein synthesis in bacteria [17-21], where the 30S subunit of the ribosome, together with the initiation factors IF1 and IF3 bind to the Translation Initiation Region (TIR) of the mRNA. This pre-initiation complex then recruits the GTP bound initiation factor IF2 and the initiating formyl-methionine tRNAfMet. Once assembled, GTP is hydrolyzed, the initiation factors are released and the 50S subunit is recruited [22] . The efficiency of translation initiation is dependent on the nucleotide sequence of the TIR, a stretch of approximately thirty nucleotides that extends from the Shine-Dalgamo region to the fifth codon of the coding sequence (i.e. the first ribosomal footprint) [23]. The TIR is the only variable element during translation. If all possible sequence permutations are considered, there are more than a quintillion TIRs (i.e. 430 >1 x 10<18>). However only a small number of TIRs are present in bacterial cells and they contain some distinctive sequence features. The most obvious is the Shine-Dalgamo (SD) sequence, a purine rich stretch of 4-9 nucleotides that hydrogen bonds with the 16S rRNA of the 30S subunit 24. This sequence guides the ribosome to the start codon, which is typically an AUG [25]. The start codon is separated from the SD sequence by a spacer region that is typically 9 nucleotides long in E. cob [26], The 5’ end of the coding sequence (~ 15 nucleotides) is also considered to be within the TIR [27] and often harbors rare codons [28,29]. Native bacterial TIRs have co-evolved with the ribosome and are less likely to form mRNA structures compared to the rest of the coding sequence [30,31]. This is thought to promote accessibility of the 30S subunit during translation initiation [28,29,32,33].

DNA constructs relating to signal peptides are known from US8361744. However, the 42 DNA constructs disclosed in US8361744 differ significantly from the DNA constructs of the present invention both with respect to DNA sequence as well as the performance of the signal peptides. Moreover, the DNA constructs of US8361744 have not been synthetically evolved and therefore do not exhibit the technical effects of the DNA constructs disclosed in the present invention.

OBJECT OF INVENTION The object of the invention is to controlling signal peptide performance.

A further object of the invention is to control recombinant protein expression via controlling signal peptide performance.

A further object of the invention is to increase (i.e. up-regulate) or to decrease (i.e. downregulate) signal peptide performance.

A further object of the invention is to provide a simple and inexpensive system of DNA constructs, expression vectors and host cells for increasing the production yields of single chain antibody fragments, hormones and other recombinant proteins.

SUMMARY OF THE INVENTION In the present invention, the inventors have solved the problem and anomaly in recombinant expression plasmid typically used to produce secreted proteins. It is in the art a common practice to place the coding sequence of the signal peptide downstream of the vector encoded 5’UTR. Hence, the resulting TIR is a fusion of the 5’UTR and the first 5 codons of the signal peptide in the TIR. The inventors hypothesized that such a TIR would not function optimally as it had not co-evolved with the ribosome. To test this hypothesis, as described in detail in the DETAILED DESCRIPTION of the present specification, the inventors synthetically evolved the TIR in the presence of host cell ribosomes. The experimental results discussed in the EXAMPLES section of the present specification clearly indicate that the performance of all signal peptides can be improved by synthetic evolution. The most striking example was PelB, which was initially the worst performing signal peptide for production of β-lactamase, but the best performing following synthetic evolution of the TIR. Thus, in summary, the performance of the signal peptide is largely coupled to the efficiency of translation initiation. The present invention provides a molecular understanding of this signal peptide performance. More importantly, the present invention provides a simple and inexpensive system comprising: DNA constructs, expression vectors, host cells, and - methods of production, for increasing the production yields of single chain antibody fragments, hormones and other recombinant proteins.

The objects of the invention are attained by the subject-matter disclosed in the claims as well as the subject-matter disclosed in the below aspects of the invention.

A first aspect of the invention relates to a DNA construct suitable for controlling signal peptide performance, wherein said DNA construct comprises: a. a Shine-Dalgamo sequence; b. an ATG start codon; c. a sequence of one of SEQ ID 1-28 comprising said ATG start codon; and d. a signal peptide encoding sequence, wherein said sequence of one of SEQ ID 1-28 comprises at least the first 9 nucleotides of said signal peptide encoding sequence.

In a preferred embodiment, said signal peptide encoding sequence comprises a sequence for expressing a signal peptide selected from MalE (maltose-binding protein precursor), OmpA (outer membrane protein A precursor), PhoA (alkaline phosphatase precursor), DsbA (thiol:disulfide interchange protein), and PelB (periplasmic pectate lyase).

In a preferred embodiment, said signal peptide encoding sequence is a sequence of one of SEQ ID 34-47.

In a preferred embodiment, said signal peptide encoding sequence expresses a signal peptide of a sequence of one of SEQ ID 29-33.

In a preferred embodiment, said Shine-Dalgamo sequences comprises nucleotide sequence TAAGAAGG in the direction of transcription.

In a preferred embodiment, said DNA construct comprise a sequence of one of SEQ ID 15-28, wherein said sequence of one of SEQ ID 15-28 comprises said Shine-Dalgamo sequence, said sequence of one of SEQ ID 1-14 and at least the first 24 nucleotides of said signal peptide encoding sequence.

In a preferred embodiment, said DNA construct is characterized in that: a sequence of one of SEQ ID 1, 2 and 3 comprises the first 9 nucleotides of a MalE signal peptide encoding sequence of one of SEQ ID 34, 35 and 36, respectively; - a sequence of one of SEQ ID 15, 16 and 17 comprises the first 24 nucleotides of a MalE signal peptide encoding sequence of one of SEQ ID 34, 35 and 36, respectively; a sequence of one SEQ ID 4, 5 and 6 comprises the first 9 nucleotides of an OmpA signal peptide encoding sequence of one of SEQ ID 37, 38 and 39, respectively; a sequence of one SEQ ID 18, 19 and 20 comprises the first 24 nucleotides of an OmpA signal peptide encoding sequence of one of SEQ ID 37, 38 and 39, respectively; a sequence of one SEQ ID 7 and 8 comprises the first 9 nucleotides of a PhoA signal peptide encoding sequence of one of SEQ ID 40 and 41, respectively; a sequence of one SEQ ID 21 and 22 comprises the first 24 nucleotides of a PhoA signal peptide encoding sequence of one of SEQ ID 40 and 41, respectively; a sequence of one SEQ ID 9, 10 and 11 comprises the first 9 nucleotides of a DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44, respectively; a sequence of one SEQ ID 23, 24 and 25 comprises the first 24 nucleotides of a DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44, respectively; - a sequence of one SEQ ID 12, 13 and 14 comprises the first 9 nucleotides of a PelB signal peptide encoding sequence of one of SEQ ID 45, 46 and 47, respectively; and/or a sequence of one SEQ ID 26, 27 and 28 comprises the first 24 nucleotides of a PelB signal peptide encoding sequence of one of SEQ ID 45, 46 and 47, respectively.

In a preferred embodiment, said DNA construct is characterized in that: said MalE signal peptide encoding sequence of one of SEQ ID 34, 35 and 36 expresses a signal peptide of a sequence of one of SEQ ID 29; said OmpA signal peptide encoding sequence of one of SEQ ID 37, 38 and 39 expresses a signal peptide of a sequence of one of SEQ ID 30; said PhoA signal peptide encoding sequence of one of SEQ ID 40 and 41 expresses a signal peptide of a sequence of one of SEQ ID 31 ; - said DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44 expresses a signal peptide of a sequence of one of SEQ ID 32; and/or said PelB signal peptide encoding sequence of one of SEQ ID 45, 46 and 47 expresses a signal peptide of a sequence of one of SEQ ID 33.

In a preferred embodiment, said DNA construct comprise a sequence of one of SEQ ID 15, 18, 21, 23 and 26.

In a preferred embodiment, said DNA construct is a synthetically evolved DNA construct.

In a preferred embodiment, said DNA construct further comprises a recombinant protein encoding sequence.

A second aspect of the invention relates to a DNA construct suitable for controlling signal peptide performance, wherein said DNA construct comprises a sequence of one of SEQ ID 15-28.

In a preferred embodiment, said DNA construct also comprises a signal peptide encoding sequence.

In a preferred embodiment, said signal peptide encoding sequence comprises a sequence for expressing a signal peptide selected from MalE (maltose-binding protein precursor), OmpA (outer membrane protein A precursor), PhoA (alkaline phosphatase precursor), DsbA (thiohdisulfide interchange protein), and PelB (periplasmic pectate lyase).

In a preferred embodiment, said sequence of one of SEQ ID 15-28 comprises the first 24 nucleotides of said signal peptide encoding sequence.

In a preferred embodiment, said signal peptide encoding sequence is a sequence of one of SEQ ID 34-47.

In a preferred embodiment, said signal peptide encoding sequence expresses a signal peptide of a sequence of one of SEQ ID 29-33.

In a preferred embodiment, said DNA construct is characterized in that: a sequence of one of SEQ ID 15, 16 and 17 comprises the first 24 nucleotides of a MalE signal peptide encoding sequence of one of SEQ ID 34, 35 and 36, respectively; a sequence of one SEQ ID 18, 19 and 20 comprises the first 24 nucleotides of an OmpA signal peptide encoding sequence of one of SEQ ID 37, 38 and 39, respectively; a sequence of one SEQ ID 21 and 22 comprises the first 24 nucleotides of a PhoA signal peptide encoding sequence of one of SEQ ID 40 and 41, respectively; a sequence of one SEQ ID 23, 24 and 25 comprises the first 24 nucleotides of a DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44, respectively; and/or a sequence of one SEQ ID 26, 27 and 28 comprises the first 24 nucleotides of a PelB signal peptide encoding sequence of one of SEQ ID 45, 46 and 47, respectively.

In a preferred embodiment, said DNA construct is characterized in that: - said MalE signal peptide encoding sequence of one of SEQ ID 34, 35 and 36 expresses a signal peptide of a sequence of one of SEQ ID 29; said OmpA signal peptide encoding sequence of one of SEQ ID 37, 38 and 39 expresses a signal peptide of a sequence of one of SEQ ID 30; said PhoA signal peptide encoding sequence of one of SEQ ID 40 and 41 expresses a signal peptide of a sequence of one of SEQ ID 31; said DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44 expresses a signal peptide of a sequence of one of SEQ ID 32; and/or said PelB signal peptide encoding sequence of one of SEQ ID 45, 46 and 47 expresses a signal peptide of a sequence of one of SEQ ID 33.

In a preferred embodiment, said DNA construct comprises a sequence of one of SEQ ID 15, 18, 21, 23 and 26.

In a preferred embodiment, said DNA construct is a synthetically evolved DNA construct.

In a preferred embodiment, said DNA construct further comprises a recombinant protein encoding sequence.

A third aspect of the invention relates to a DNA construct suitable for controlling signal peptide performance, wherein said DNA construct comprises a sequence of one of SEQ ID 49, 51, 53, 55 and 57.

In a preferred embodiment, said DNA construct also comprises a signal peptide encoding sequence.

In a preferred embodiment, said signal peptide encoding sequence comprises a sequence for expressing a signal peptide selected from MalE (maltose-binding protein precursor), OmpA (outer membrane protein A precursor), PhoA (alkaline phosphatase precursor), DsbA (thiol:disulfide interchange protein), and Pelb (periplasmic pectate lyase).

In a preferred embodiment, said signal peptide encoding sequence is a sequence of one of SEQ ID 58-62.

In a preferred embodiment, said signal peptide encoding sequence expresses a signal peptide of a sequence of one of SEQ ID 29-33.

In a preferred embodiment, said DNA construct is characterized in that: - a sequence of SEQ ID 49 comprises the first 24 nucleotides of a MalE signal peptide encoding sequence of SEQ ID 58; a sequence of SEQ ID 51 comprises the first 24 nucleotides of an OmpA signal peptide encoding sequence of SEQ ID 59; a sequence of SEQ ID 53 comprises the first 24 nucleotides of a PhoA signal peptide encoding sequence of SEQ ID 60; a sequence of SEQ ID 55 comprises the first 24 nucleotides of a DsbA signal peptide encoding sequence of SEQ ID 61; and/or a sequence of SEQ ID 57 comprises the first 24 nucleotides of a PelB signal peptide encoding sequence of SEQ ID 62.

In a preferred embodiment, said DNA construct is characterized in that: said MalE signal peptide encoding sequence of SEQ ID 58 expresses a signal peptide of sequence of SEQ ID 29; said OmpA signal peptide encoding sequence of SEQ ID 59 expresses a signal peptide of sequence of SEQ ID 30; - said PhoA signal peptide encoding sequence of SEQ ID 60 expresses a signal peptide of sequence of SEQ ID 31; said DsbA signal peptide encoding sequence of SEQ ID 61 expresses a signal peptide of sequence SEQ ID 32; and/or said PelB signal peptide encoding sequence of SEQ ID 62 expresses a signal peptide of a sequence of one of SEQ ID 3.

In a preferred embodiment, said DNA construct further comprises a recombinant protein encoding sequence.

A fourth aspect of the invention relates to an expression vector comprising a DNA construct according to the above disclosed first, second or third aspects of the invention, wherein the expression vector is preferably a plasmid, more preferably PET expression vector, and most preferably pet28A A fifth aspect of the invention relates to a host cell comprising the above disclosed expression vector of the fourth aspect of the invention, wherein said host cell is preferably a bacterial cell, more preferably said bacterial cell is E. coli and most preferably E. coli strain BL21(DE3) pLysS.

A sixth aspect of the invention relates to a recombinant protein expressed by the above disclosed host cell of the fifth aspect of the invention.

A seventh aspect of the invention relates to a method of expressing the above disclosed recombinant protein of the sixth aspect of the invention, said method comprising the steps of: introducing said DNA construct according to the above disclosed first, second or third aspects of the invention into an expression vector; introducing the expression into a host cell; growing the host cell; and and recovering the recombinant protein from the host cell.

An eighth aspect of the invention relates to an RNA molecule expressed by a DNA construct according to the above disclosed first, second or third aspects of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. A comparison of commonly used signal peptides. (A) An overview of the expression cassettes used in this experiment. The TIR region represented by the boxed area, from the Shine-Dalgamo to the fifth codon of the signal sequence. The coding sequences for five commonly used signal peptides (MalE, OmpA, PhoA, DsbA, PelB) were cloned into the pET28a vector, upstream of the mature coding sequences for β-lactamase, scFvor FtYfgM<45-170>. Protein production was induced for two hours, then a volume of cells corresponding to 0.2 OD6oo units of cells were harvested, separated by a 12 % SDS-PAGE and protein levels determined by immuno-blotting with antisera to β-lactamase (B), or the poly-Histidine tag of scFv(C) and FtYfgM<45-170 >(D). To ensure that protein loading was consistent between the samples, the membranes were stained with Amido black after immunodetection. ‘Pre’ denotes the precursor form of the protein, which contains the signal sequence and is presumed to be in the cytoplasm. ‘Mat’ precursor denotes the mature form, which is presumed to be in the periplasm as the signal peptide has been cleaved.

Figure 2. Improved signal peptide performance following synthetic evolution of the TIR (A) A synthetic evolution approach was used to convert a TIRto a TIR_ The TIR is defined as the region from the Shine-Dalgamo (half-dome) to codon 5 of the signal peptide. (B) mRNA has a high propensity to form structures, thus a TIRcan be sequestered into short- (top) or long-range structures (middle). Synthetic evolution should select a TIRthat is relaxed and more accessible to the ribosome (bottom). (C) An overview of the synthetic evolution process. A TIRwas constructed by completely randomising the six nucleotides immediately upstream of the AUG start codon, and partially randomising the six nucleotides immediately downstream of the AUG start codon (allowing synonymous codons changes only). The TIRwas transformed into E. coli BF21(DE3) pLysS and plated on increasing concentrations of ampicillin. A TIRwas identified on the plate containing the highest concentration of ampicillin relative to the TIRvariant. (D) β-lactamase production levels from TIR/ TIRpairs were assessed by immuno-blotting. In this experiment, β-lactamase production was induced for two hours, then a volume of cells corresponding to 0.2 OD600 units of cells were harvested, separated by a 12 % SDS-PAGE and protein levels were determined by immuno-blotting with antisera to β-lactamase. To ensure that protein loading was consistent between the samples, the membrane was stained with Amido black after immuno-detection. ‘Pre’ denotes the precursor form of the protein, which contains the signal peptide fused version of β-lactamase, which we presume to be in the cytoplasm as the signal peptide is still present. ‘Mat’ precursor denotes the mature version of β-lactamase, which presumably is in the periplasm as the signal peptide has been cleaved. (E) β-lactamase activity from TIR/ TIRpairs was assessed using the disc diffusion assay. Here a filter disc containing 2 mg of ampicillin was placed on top of an LB -agar plate containing a lawn of bacteria expressing β-lactamase from either a -TIRor a TIR. The diameter of the growth-inhibition zone was measured for each experiment. In all cases, a TIRconferred more resistant to ampicillin than -TIR.

Figure 3. Time-course analysis of β-lactamase production. (A) An illustration of the experimental workflow used. (B) At each time point, a volume of cells was extracted, then separated by SDS-PAGE and immuno-blotted with antisera to β-lactamase. Band intensities were obtained from immuno-blots by densitometric analysis and normalised to the highestvalue.

Figure 4. A synthetically evolved TIR (TIR) is transferable. (A) Expression levels of ScFvand FtYfgM<45-170 >using five different signal peptides. In each instance TIR/ TIRpairs were assessed by immuno -blotting. The TIRhad originally been selected for β-lactamase (see Figure 2). In this experiment, protein production was induced for two hours, then a volume of cells corresponding to 0.2 OD6oo units of cells were harvested, separated by a 12 % SDS-PAGE and protein levels were determined by immunoblotting with antisera to a poly-histidine tag. ‘Pre’ denotes the precursor form of the protein and ‘Mat’ denotes the mature version.

Figure 5. Production and purification of the human growth hormone (hGH) using a TIR. (A) Production levels of hGH using five different signal peptides. In each instance the difference between TIR/ TIRpairs were assessed by immuno -blotting. The TIRhad originally been selected for β-lactamase (see Figure 2). In this experiment, protein production was induced for two hours, then a volume of cells corresponding to 0.2 OD6oo units of cells were harvested, separated by a 12 % SDS-PAGE and protein levels were determined by immuno -blotting with antisera to a poly-histidine tag. ‘Pre’ denotes the precursor form of the protein and ‘Mat’ denotes the mature version. (B) An overview of the methodology used to purify hGH. (C) Analysis of the purified hGH by Size-Exclusion Chromatography (SEC). (D) Purified hGH was analysed by SDS-PAGE under denaturing- and non-denaturing conditions. (E) Activity of the purified hGH by using the MTS cell proliferation assay.

DETAILED DESCRIPTION The present invention relates to controlling signal peptide performance with a DNA construct wherein the DNA construct comprises: e. a Shine-Dalgamo sequence, f. an ATG start codon, g. a sequence of one of SEQ ID 1-28 comprising said ATG start codon, and h. a signal peptide encoding sequence, wherein the sequence of one of SEQ ID 1-28 comprises at least the first 9 nucleotides of the signal peptide encoding sequence. When the DNA construct comprise a sequence of one of SEQ ID 15-28 then such a sequence comprises the Shine-Dalgamo sequence, said sequence of one of SEQ ID 1-14 and at least the first 24 nucleotides of the signal peptide encoding sequence.

The signal peptide encoding sequence may comprises a sequence for expressing a signal peptide selected from MalE, OmpA, PhoA, DsbA and PelB. A specific signal peptide encoding sequence may be a sequence of one of SEQ ID 34-47 which may express a signal peptide of a sequence of one of SEQ ID 29-33 indicated in Table 1.

However, DNA constructs comprising a sequence of one of SEQ ID 48-57 may also be used.

The preferred combinations of (a) a DNA construct sequence, (b) a signal peptide peptide sequence, and/or (c) a signal peptide DNA sequence, are disclosed in Tables 1 and 2.

Table 1. Signal peptides and their corresponding peptide and DNA sequences. Several peptide DNA sequences may express the same peptide sequence due to the synonymous codon changes discussed in Example 2, Figure 2 and elsewhere in the specification.

Image available on "Original document" Image available on "Original document" Image available on "Original document" The invention further relates to an expression vector comprising the above-mentioned DNA construct. Additionally, the present invention relates to host cell comprising said expression vector. Furthermore, the present invention relates to a recombinant protein expressed by said host cell as well as a method for expressing said recombinant protein. The DNA construct may further comprise a recombinant protein encoding sequence.

The above described DNA constructs, expression vectors, host cell and recombinant proteins have been described in the EXAMPLES and EXPERIMENTAL PROCEDURES sections of this specification. Moreover, the results of the comparative tests are discussed in the EXAMPLES section to provide evidence of the increased (i.e. up-regulated) signal peptide performance of DNA constructs comprising a sequence of one of SEQ ID 1-28. However, the present invention may alternatively be used for decreasing the signal peptide performance of DNA constructs comprising a sequence of one of SEQ ID 48-57; such an effect may be relevant in cases when the expression of recombinant protein needs to be down-regulated.

Some of the significant comparative tests discussed in the EXAMPLES are summarized in the following paragraphs before the EXAMPLES section.

As already indicated, the present invention relates to improving signal peptide performance by synthetically evolving the TIR. The present invention further provides a simple and inexpensive solution for increasing the production yields of secreted proteins in bacterial cell factories. Moreover, the present invention will be compatible with other published methods; such as those that use titratable promoters to tune transcription rates of secreted proteins [40]. A potential problem is the need for screening of large TIR. However, in the present invention, said problem was solved by using β-lactamase protein, which confers resistance to β-lactam antibiotics and can be easily screened; this embodiment of the present invention is discussed in detail in Examples 2 and 3. For proteins where no simple screening assay is available it is possible to translationally-couple β-lactamase to the recombinant protein and thereby solve potential problems. It is also possible to use the signal peptides in pET28a vectors from the present invention, which possess a TIRand which improved production yields of a single chain antibody fragment, a hormone and another recombinant protein in Escherichia cob; this embodiment of the invention is discussed in detail in Example 4 A link between signal peptide performance and the efficiency of translation initiation has been implied previously. Punginelli and co-workers noted that non- synonymous nucleotide changes in the signal peptide of the Tat-dependent formate dehydrogenase increased production levels by up to 60-fold in E. coli [38]. And Ng and Sarkar noted that synonymous changes to the Usp45sp signal peptide in Lactococcus lactis helped to increase production levels of a nuclease and an amylase by approximately 15% [39]. Both studies postulated that the nucleotide changes helped to relax mRNA structure that had sequestered the TIR.

The present invention also demonstrates that nucleotide changes in the TIR can influence production of secreted proteins (although this could not be correlated to changes in mRNA structure). Significantly, the present invention goes beyond the current literature as indicated in the comparative experiments described in Example 2 and Figure 2 which demonstrate that signal peptides generally under-perform in protein production experiments because the TIR, encompassing the 5’ UTR of the plasmid and the 5’ terminus of the gene coding sequence, has not co-evolved with the ribosomes of the host cell.

As further disclosed in Example 2 and Figure 2, the inventors were able to support this molecular explanation by demonstrating that a synthetic evolution process could improve the performance of all commonly used signal peptides. As indicated earlier in the specification, the most striking example was PelB, which was initially the worst performing signal peptide for production of β-lactamase, but the best performing following synthetic evolution of the TIR as illustrated in Figure 2D. Thus, the performance of the signal peptide is largely coupled to the efficiency of translation initiation.

EXAMPFES The following examples are not to be interpreted as limiting the scope of the invention. For experimental details pertaining to the examples below, the skilled reader is directed to the separate EXPERIMENTAL PROCEDURES section below.

Example 1 - Production of periplasmic proteins with commonly used signal peptides Five signal peptides that are commonly used for the production of recombinant proteins in the periplasm of E. cob were selected (MalE, OmpA, PhoA, DsbAand PelB; see Table 1; see SEQ-ID 29-33). The coding sequences for these signal peptides were cloned into the commonly used pET28a expression plasmid, upstream of the coding sequence for β-lactamase (Figure 1A). To determine how efficiently the signal peptides supported the synthesis and secretion of β-lactamase the expression plasmids were transformed into the E. coli strain BL21(DE3) pLysS and a mild induction protocol was used to initiate transcription (0.05 mM IPTG for 2 hours at 30 °C). Following the induction period, whole cells were collected, and proteins were separated by SDS-PAGE and immuno -blotted, so that the secreted (Mature) and non-secreted (Precursor) β-lactamase could be distinguished. The experiment indicated that there were large differences in production levels (Figure 1B). MalE, OmpA, PhoA, DsbAsupported a comparatively high-level of β-lactamase production, whereas PelBdid not. The experiment also indicated that there were significant differences in secretion efficiency between the different signal peptides. MalEand PelBwere effective in supporting the secretion of β-lactamase to the periplasm, whereas OmpA, PhoAand DsbAwere deemed less effective as there was a prominent precursor band.

To evaluate the performance of the signal peptides with other recombinant proteins, they were fused to a single chain variable fragment that recognizes the human epidermal growth factor receptor protein 2 protein (scFv) and a soluble fragment of the periplasmic chaperone YfgM from Francisella tularensis (FtYfgM<45-170>). Again, there were considerable differences in production levels across the different signal peptides (Figure 1C and D). Moreover, there were considerable differences between β-lactamase, scFvand FtYfgM<45-170 >(Figure 1B vs C vs D). Taken together, these observations demonstrate that signal peptide performance is varied and unpredictable during the synthesis and secretion of recombinant periplasmic proteins. This conclusion is supported by a large body of published work, but a molecular explanation for the phenomenon remains elusive [3].

Example 2 - Signal peptide performance is coupled to translation initiation The expression plasmids used in the previous experiments had been assembled by genetically sandwiching the nucleotide sequence encoding the signal peptide between the vector encoded 5’UTR and the 5’ end of the mature coding sequence for β-lactamase, scFvor FtYfgM45-170 (Figure 2A). Each expression plasmid therefore contained a different TIR (Table 2). The inventors hypothesized that these TIRs might not be optimal for translation initiation as they had not co-evolved with the host cell ribosomes, possibly leading to unfavorable interactions at the mRNA level (Figure 2B). They are therefore referred to as a TIR.

Synthetic (or directed) evolution was used to select TIRs that were more compatible with the host cell ribosomes. In the experiment, TIRwere created from expression plasmids containing the MalE, OmpA, PhoA, DsbAand PelBfused to β-lactamase. In the design of the TIR, the six nucleotides immediately upstream from the AUG start codon were completely randomized, and the six nucleotides immediately downstream from the AUG start codon were randomized with synonymous codon changes only (Figure 2C) [34,35]. Each TIRtheoretically contained >18,000 expression plasmids with a different TIR. The TIRwere transformed into BF21(DE3) pFysS and plated onto LB agar containing 0.05 mM IPTG and increasing concentrations of ampicillin (Figure 2C). A colony that was resistant to a high concentration of ampicillin was selected, the expression plasmid was isolated and the TIR sequenced. These TIRs as referred to as synthetically evolved (TIR-) (Table 2) Table 2. Nucleotide sequences of the TIRand corresponding TIRused in this study. The TIR is defined as the region from the Shine-Dalgarno to codon five of the signal peptide.

Image available on "Original document" Image available on "Original document" Underlined region was randomised during the synthetic evolution process <2>Nucleotides marked in bold text were changed in TIR- <3>SEQ ID is indicated for underlined region (referred to as “short sequence” in the sequence listing) and full nucleotide sequence (referred to as “full sequence” in the sequence listing), respectively Expression plasmids containing either a TIRor TIR-were retransformed into BL21(DE3) pLysS and the production levels of β-lactamase compared by immuno -blotting. After a two-hour induction period we observed that the production levels of periplasmic β-lactamase were significantly higher when using a TIR-compared to the TIR(Figure 2D). Note that production of β-lactamase from each TIRwas undetectable on these blots because the difference with the TIR-was too large to capture at this time point (see below). Consistent with this observation, disc diffusion assays confirmed that the TIR-supported a higher level of resistance to ampicillin than the TIR(Figure 2E). Figures 2E and 2D illustrate comparative experiments using TIRand TIR-wherein (i) TIR-comprised SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRcomprised SEQ ID of 48, 50, 52, 54 and 56, respectively.

The inventors speculate that the difference in production levels from the TIR/ TIR-pairs was a result of mRNA relaxation, but the inventors were unable to support this speculation by using mRNA fold prediction programs. The lack of a correlation could reflect the fact that (1) mRNA relaxation is not the sole determinant, (2) mRNA structure is notoriously difficult to predict, and/or (2) existing algorithms only handle short stretches of nucleotides (not an entire mRNA). Nevertheless, the experiment does demonstrate that all signal peptides were under-performing when a TIRwas used. And significantly, the performance of all signal peptides could be improved by synthetically evolving the TIR. This phenomenon was most easily seen with PelB, which gave the lowest levels of β-lactamase production when expressed from a TIR(Figure 1B) but the highest when expressed from a TIR(Figure 2E). Synthetic evolution of the TIR had therefore ‘converted’ the PelBfrom a ‘poor-performing’ signal peptide to a ‘topperforming’ signal peptide without changing a single amino acid. The data therefore demonstrate that signal peptide performance is tightly coupled to translation initiation in bacterial cell factories.

Example 3 - Production of recombinant periplasmic proteins using a TIR In the previous series of experiments a mild induction protocol had been used (0.05 mM IPTG for 2 hours at 30 °C), so that differences in protein production could be assessed in the absence of a metabolic load on the cell. The concern about metabolic load largely relates to the Sec translocon, which is believed to be a bottleneck in the production of periplasmic proteins [36,37], When production levels of periplasmic proteins are too high, the translocon could become saturated and the recombinant protein may be retained in the cytoplasm. To determine if expression plasmids with a TIRwould saturate the Sec translocon, the inventors induced with either a low (0.05 mM) or a high (0.5 mM) IPTG concentration and monitored production over a 5-hour period (Figure 3A). It was observed that, at all but one time-point, a TIRproduced more periplasmic β-lactamase than the corresponding TIR(Figure 3B). This observation was made at both low and high concentrations of IPTG. These time-course experiments therefore indicated that the Sec translocon was able to cope with the increased production levels that were reached using a TIRfigures 3B illustrates comparative experiment using TIRand TIRwherein (i) TIRcomprised SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRcomprised SEQ ID of 48, 50, 52, 54 and 56, respectively.

Example 4 - Using TIRas a generic solution In this set of experiments the coding sequences of scFvHER2 and FtYfgM<45-170 >were expressed as fusions to the original five signal peptides, using both the TIRand TIREVOLVED pairs. The expression plasmids were again transformed into BL21(DE3) pLysS and production was monitored using a mild induction protocol (0.05 mM IPTG for 2 hours at 30 °C). As we had observed for β-lactamase, the TIRalways produced more protein than the corresponding TIR(Figure 4). It was noted that signal peptide performance was varied; the most effective signal peptide for production of scFvwas PhoA, whilst the most effective for FtYfgM<45-170 >was MalE. Thus, signal peptide performance might partly be explained by compatibility of the signal peptide. Figure 4 illustrates comparative experiments using TIRand TIRwherein (i) TIRcomprised SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRcomprised SEQ ID of 48, 50, 52, 54 and 56, respectively.

A similar approach was taken to produce the human growth hormone (hGH). Here we observed that the most effective TIRfor production of hGH was the one coupled to the PelB(Figure 5A). To assess how much more protein was produced the N-terminally His-tagged hGH was purified by Immobilized Metal Affinity Chromatography (IMAC), the His-tag removed by proteolytic processing, and the sample polished by Size Exclusion Chromatography (SEC) (Figure 5B). The yield of purified hGH was more than 3 -fold higher using the TIRcompared to the TIR(2.56 mg/L vs 0.79 mg/L).

Importantly, we could not detect any difference in the quality of the purified hGH, as judged by monodispersity of the sample following SEC (Figure 5C), the proportion of protein that had formed disulphide bonds (Figure 5D), or the activity of the protein when tested by the MTS cell proliferation assay (Figure 5E). Figures 5 A, 5C, 5D and 5E illustrate comparative experiments using TIRand TIRwherein (i) TIRcomprised SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRcomprised SEQ ID of 48, 50, 52, 54 and 56, respectively.

Taken together, this series of experiments indicate that the pET28a-based vectors containing signal peptides with a TIRcan be used as a generic solution to increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coli without compromising protein quality.

EXPERIMENTAL PROCEDURES Molecular cloning The sequences encoding MalE, OmpA, PhoA, DsbA, PelB, β-lactamase, hGH and FtYfgM<45-170 >were chemically synthesised (Genscript, USA). The sequence encoding scFvwas obtained from the pHP2-15 plasmid [44], To generate expression clones, the coding sequences and the pET28a vector were amplified by PCR using the Q5 polymerase (New England Biolabs, UK). The coding sequences were then cloned between the Ncol and Ndel restriction enzyme sites using the Gibson cloning method. Enzymes used for Gibson cloning were obtained from New England Biolabs, UK.

Synthetic evolution of the TIR TIRLIBRARIES were generated by amplifying the expression plasmids by PCR, using overlapping primers as previously described [34,35]. The forward primer was approximately 45 nucleotides in length and was partly degenerate. The design enabled complete randomization of the six nucleotides upstream of the AUG start codon, and partial randomization of the six nucleotides downstream stream of the AUG start codon (synonymous codons only). The reverse primer was always the same sequence (5’-CTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATC-3’). It overlapped with the forward primer by 13 nucleotides thus allowing circularization of the PCR product by homologous recombination in E. coli MC1061. The PCR was carried out using the Q5 polymerase (New England Biolabs, UK) in a program that consisted of 94 °C for 5 min and then 30 cycles of 95 °C for 45 s, 48-68 °C for 45 s (using a gradient thermocycler), 72 °C for 6 min and a final elongation step of 72 °C for 5 min. Specific PCR products that were amplified at the lowest annealing temperature were treated with Dpnl, then transformed into chemically competent E. coli MC1061. The transformation was seeded into 100 mL of Luria-Bertani containing 50 μg/mL kanamycin and incubated overnight at 37 °C. Isolation of the TIRwas carried out using ten E.N.Z.A DNA mini kit purification columns (Omega Biotek, USA) and pooling of the eluates.

TIRwere screened by transforming chemically competent BL21(DE3) pLysS and identifying clones that survived on the highest concentration of ampicillin. Here 0.5 pg of the TIRwas transformed into 50 pL of chemically competent BL21(DE3) pLysS using standard protocols. The entire transformation was then seeded into 3 mL of LB containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol. Cultures were grown at 37 °C with shaking for 16 h. Cultures were then back-diluted (1:50) into 5 mL of LB containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol and incubated as before until an OD600 of ~0.3 was reached. Expression of the coding sequence was induced by streaking a volume of cells corresponding to 0.002 OD600 units on LB agar containing 0.05 mM isopropyl-β-D thiogalactopyranoside (IPTG) and increasing concentrations of ampicillin (100-5000 μg/mL).

Note that kanamycin and chloramphenicol were omitted from the plates. The plates were then incubated for 16 h at 37 °C. Colonies formed at higher ampicillin concentrations were selected for further analysis and sequencing (Euro fins MWG operon, Germany).

Immuno-blotting Cultures were grown at 37 °C with shaking for 16 h, then back-diluted (1:50) into 5 mL of LB containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol and incubated as before until an OD600 of -0.3-0.5 was reached. Expression of the coding sequence was induced with 0.05 mM IPTG for 2 h at 30 °C. A volume of cells corresponding to an OD600 of either 0.02 or 0.2 was harvested by centrifugation then resuspended in 2x Laemlli loading buffer [125 mM Tris-HC1 pH 6.8, 4% SDS, 3% Glycerol, 0.02% bromophenol blue, 20% β-mercaptoethanol]. Proteins were separated by 12% SDS-PAGE then transferred to a nitrocellulose membrane using a semi-dry transfer apparatus (Bio-Rad, USA). The nitrocellulose membranes were probed with an antibody against either β-lactamase (Thermo Scientific, USA) or the polyhistidine tag (His-Probe, ThermoFisher Scientific, USA). Binding was detected using antimouse IgG linked to horseradish peroxidase (GE healthcare, USA) and a SuperS ignal West femto luminol/enhancer solution (ThermoFisher Scientific, USA). Luminescence emitting from the nitrocellulose membrane was detected using an Azure Biosystems c600 device.

Disc diffusion assays Cells were grown in LB containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol until an OD600 of -0.3. A volume of cells corresponding to an OD600 of 0.002 was then plated onto LB agar (lacking all antibiotics). A sterile filter disc containing 2 mg ampicillin was then placed on top of the cells and the plates were incubated at 37 °C for 16 h. Zones of growth inhibition were measured using a standard ruler.

Purification of hGH Expression plasmids harboring pET28a pelB-hGH were transformed into the expression host BL21(DE3) pLysS and grown on LB agar plates containing 50 μg/mL kanamycin and 34 pg/mL chloramphenicol. Single colonies were used to inoculate 100 mL of LB plus antibiotics medium which was grown overnight at 37 °C with shaking at 180 RPM. Overnight pre-cultures were used to inoculate 2 L flasks containing 1 L of LB media plus antibiotics, to a starting OD600 of 0.05. Cultures were grown to an OD600 of 0.7, at which point, flasks were incubated on ice for 10 minutes. Induction proceeded with the addition of 0.01 mM IPTG and incubation for 16 hours at 18 °C with shaking at 180 RPM. Cells were harvested for 20 minutes at 4,000 x g. Cell pellets were resuspended in 50 mL suspension buffer (50 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole pH 8.0 and lx protease inhibitor cocktail (cOmplete, Roche, USA)). Cell suspensions were homogenized with a glass Dounce homogenizer followed by cell disruption using an Avestin emulsiflex C3 high-pressure homogenizer (Avestin, Canada). Cell debris was removed by centrifugation at 20,000 x g for 30 minutes. Samples were applied to 2.5 mL Ni-sepharose (GE Healthcare) and batch incubated at 4 °C for one hour on a benchtop roller. The column was washed with 20 column volumes (50 mL) of wash buffer (50 mM Tris pH 8.0, 500 mM NaCl and 50 mM imidazole pH 8.0), followed by elution with 30 mL of elution buffer (50 mM Tris pH 8.0, 500 mM NaCl and 500 mM imidazole pH 8.0). The elution fraction was concentrated and buffer exchanged (50 mM Tris pH 8.0, 150 mM NaCl and 20 mM imidazole) using a centrifugal filter with a nominal MWCO of 10 kDa (Amicon, Merck Millipore). The N-terminal his-tag was proteolytically removed with TEV protease (purified in-house) at a 1:10 weight ratio and allowed to incubate overnight at 4 °C. Samples were reverse Ni purified, concentrated and applied to size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare, Sweden) in 50 mM Tris pH 8.0 and 100 mM NaCl. Relevant fractions were pooled, and concentrated. Sample concentration was measured by the BCA protein assay kit (Pierce, ThermoFisher Scientific, USA) and protein quality assessed by SDS-PAGE.

Calculation of final yield per liter was determined by accounting of final volume, final OD at the conclusion of expression, and final concentration of purified hGH.

MTS cell proliferation assay The breast cancer MCF7 cell line (ATCC) was maintained in RPMI-1640 medium containing 10% FBS, 2mM glutamine and 1% penicillin streptomycin (Gibco/Thermo Fisher Scientific) at 37 °C in a humidified atmosphere at 5% CO2. Cell proliferation following titration of purified hGH was determined according to the CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay (MTS) protocol (Promega). Briefly, 1x10<4 >MCF7 cells were seeded in triplicate, in 100 μL aliquots into 96 well plates, followed by serum starvation for 24 hours, prior to commencing the proliferation assay. Serially diluted hGH was added to the medium at a final concentration ranging from 0 to 400 ng/mL. Cell proliferation was assessed after 48 hours of incubation, by addition of MTS and the electron coupling reagent PMS. The conversion of MTS to formazan was measured by absorbance at 490 nm using a SpectraMax plate reader. Background absorbance was corrected by subtraction of wells containing RPMI. hGH EC50 was calculated using GraphPad Prism 8.1.0.

Claims (19)

1. DNA construct suitable for regulating signal peptide performance, Wherein said DNAconstruct comprises:a. a Shine-Dalgamo sequence;b. an ATG start codon;c. a sequence of one of SEQ ID l-28 comprising said ATG start codon; andd. a signal peptide encoding sequence,Wherein said sequence of one of SEQ ID l-28 comprises at least the first 9 nucleotides of said signal peptide encoding sequence.

2. DNA construct according to claim l, Wherein said signal peptide encoding sequencecomprises a sequence for eXpressing a signal peptide selected from MalE (maltose-binding protein precursor), OmpA (outer membrane protein A precursor), PhoA(alkaline phosphatase precursor), DsbA (thiolzdisulfide interchange protein), and Pelb (periplasmic pectate lyase).

3. DNA construct according to claim l or 2, Wherein said signal peptide encoding sequence is a sequence of one of SEQ ID 34-47.

4. DNA construct according to any one of the previous claims, Wherein said signalpeptide encoding sequence eXpresses a signal peptide of a sequence of one of SEQ ID 29-33.

5. DNA construct according to any one of the previous claims, Wherein said Shine-Dalgarno sequences comprises nucleotide sequence TAAGAAGG in the direction of transcription.

6. DNA construct according to any one of the previous claims, Wherein said DNAconstruct comprise a sequence of one of SEQ ID 15-28, Wherein said sequence of oneof SEQ ID l5-28 comprises said Shine-Dalgamo sequence, said sequence of one ofSEQ ID l-l4 and at least the first 24 nucleotides of said signal peptide encoding SCqUCnCC .

7. DNA construct according to any one of the previous c1aims, Wherein: a sequence of one of SEQ ID 1, 2 and 3 comprises the first 9 nuc1eotides ofa Ma1E signal peptide encoding sequence of one of SEQ ID 34, 35 and 36,respective1y; a sequence of one of SEQ ID 15, 16 and 17 comprises the first 24nuc1eotides of a Ma1E signal peptide encoding sequence of one of SEQ ID34, 35 and 36, respective1y; a sequence of one SEQ ID 4, 5 and 6 comprises the first 9 nuc1eotides of anOmpA signa1 peptide encoding sequence of one of SEQ ID 37, 38 and 39,respective1y; a sequence of one SEQ ID 18, 19 and 20 comprises the first 24 nuc1eotidesof an OmpA signa1 peptide encoding sequence of one of SEQ ID 37, 38and 39, respective1y; a sequence of one SEQ ID 7 and 8 comprises the first 9 nuc1eotides of aPhoA signa1 peptide encoding sequence of one of SEQ ID 40 and 41,respective1y; a sequence of one SEQ ID 21 and 22 comprises the first 24 nuc1eotides of aPhoA signa1 peptide encoding sequence of one of SEQ ID 40 and 41,respective1y; a sequence of one SEQ ID 9, 10 and 11 comprises the first 9 nuc1eotides ofa DsbA signa1 peptide encoding sequence of one of SEQ ID 42, 43 and 44,respective1y; a sequence of one SEQ ID 23, 24 and 25 comprises the first 24 nuc1eotidesof a DsbA signa1 peptide encoding sequence of one of SEQ ID 42, 43 and44, respective1y; a sequence of one SEQ ID 12, 13 and 14 comprises the first 9 nuc1eotidesof a Pe1B signa1 peptide encoding sequence of one of SEQ ID 45, 46 and47, respective1y; and/or a sequence of one SEQ ID 26, 27 and 28 comprises the first 24 nuc1eotidesof a Pe1B signa1 peptide encoding sequence of one of SEQ ID 45, 46 and47, respectively.

8. DNA construct according to the previous c1aim, Wherein: - said MalE signal peptide encoding sequence of one of SEQ ID 34, 35 and36 eXpresses a signal peptide of a sequence of one of SEQ ID 29; - said OmpA signal peptide encoding sequence of one of SEQ ID 37, 38 and39 eXpresses a signal peptide of a sequence of one of SEQ ID 30; - said PhoA signal peptide encoding sequence of one of SEQ ID 40 and 41eXpresses a signal peptide of a sequence of one of SEQ ID 31; - said DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and44 eXpresses a signal peptide of a sequence of one of SEQ ID 32; and/or - said PelB signal peptide encoding sequence of one of SEQ ID 45, 46 and47 eXpresses a signal peptide of a sequence of one of SEQ ID 33.

9. DNA construct according to any one of the previous claims, Wherein said DNA construct comprise a sequence of one of SEQ ID 15, 18, 21, 23 and 26.

10. DNA construct according to any one of the previous claims, Wherein said DNA construct is a synthetically evolved DNA construct.

11. DNA construct according to any one of the previous claims, Wherein said DNA construct further comprises a recombinant protein encoding sequence.

12. DNA construct according to the previous claim, Wherein said signal peptide encodingsequence is operably linked to said recombinant protein encoding sequence in the direction of transcription.

13. EXpression vector comprising the DNA construct according to any one of claims 1-12,Wherein the eXpression vector is preferably a plasmid, more preferably PET eXpression vector, and most preferably pet28A

14. Host cell comprising the vector according to claim 13, Wherein said host cell ispreferably a bacterial cell, more preferably said bacterial cell is E. coli and most preferably E. coli strain BL21(DE3) pLysS.

15. Recombinant protein expressed by the host cell according to claim 14.

16. Method of eXpressing a recombinant protein according to claim 15, comprising thesteps of: a. introducing said DNA construct according to any one of the claims 1-12 intoan expression vector; b. introducing the eXpression into a host cell;growing the host cell; and d. and recovering the recombinant protein from the host cell.

17. RNA expressed by a DNA construct according to any one of the claims 1-12.

18. Use of a DNA construct according to any one of the claims 1-12 for regulating signal peptide performance.

19. Use of a DNA construct according to claims 18 for regulating signal peptideperformance, Wherein said regulating signal peptide performance is up-regulating signal peptide performance.