SE543946C2 - 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 SIGNALPEPTIDE PERFORMANCE AS WELL AS VECTORS, HOST CELLS ANDRECOMBINANT 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 pharrnaceutical industries for theproduction of high-value recombinant proteins. Classic examples include industrial enzymes,horrnones and antibody fragments, Which generate billions of dollars in revenue annually[l,2]. These recombinant proteins are typically engineered With an N-terrninal signal peptideso that they Will be secreted out of the bacterial cytoplasm [3]. For industrial enzymes, Whichare usually produced in gram-positive bacteria such as Bacillus subtilis, secretion from thecytoplasm to the culture supernatant simplifies purification and doWnstream processing. Forhorrnones and antibody fragments, Which are usually produced in gram-negative bacteria likeEscherichia coli, secretion from the cytoplasm to the oxidizing environment of the periplasmis 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 cytoplasmicmembrane, and secretes proteins to the envelope and beyond. Secreted proteins are typicallytargeted to Sec by an N-terminal signal peptide. Signal peptides vary in length and amino acidsequence, 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 typicallycontains the signal peptidase recognition site (Ala-X-Ala) [8,9]. They also have a distinctivecodon usage, Which includes a biased use of the AAA (Lys) codon at the second position, anda high frequency of non-optimal codons [l0-l4]. 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 [l5]. Upon arrival at Sec the signal peptide also promotes binding to the SecAchaperone, thereby allosterically activating Sec for protein secretion [l6]. Given thesemultiple 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 forone recombinant protein often supports a loW-level of protein synthesis and secretion foranother. Herein We refer to this phenomenon as signal peptide performance (i.e. signal peptidestrength). Since it is not possible to predict how Well a signal peptide Will perform With agiven recombinant protein, it is common practice to screen large signal peptide-libraries forone that supports a high-level of protein synthesis and secretion [3]. This approach is bothtime-consuming and expensive. Hence, there is a need for a molecular understanding of signalpeptide performance since as it could lead to new methods for (l) identifying suitable signalpeptides, 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 [l7-2l], Where the30S subunit of the ribosome, together With the initiation factors IFl and IF3 bind to theTranslation Initiation Region (TIR) of the mRNA. This pre-initiation complex then recruitsthe GTP bound initiation factor IF2 and the initiating formyl-methionine tRNAfMet. Onceassembled, GTP is hydrolyzed, the initiation factors are released and the SOS subunit isrecruited [22]. The efficiency of translation initiation is dependent on the nucleotide sequenceof the TIR, a stretch of approximately thirty nucleotides that extends from the Shine-Dalgarnoregion to the fifth codon of the coding sequence (i.e. the first ribosomal footprint) [23]. TheTIR is the only variable element during translation. If all possible sequence permutations areconsidered, there are more than a quintillion TIRs (i.e. 430 >l x 1018). HoWever only a smallnumber of TIRs are present in bacterial cells and they contain some distinctive sequencefeatures. The most obvious is the Shine-Dalgamo (SD) sequence, a purine rich stretch of 4-9nucleotides that hydrogen bonds With the l6S rRNA of the 30S subunit 24. This sequenceguides the ribosome to the start codon, Which is typically an AUG [25]. The start codon isseparated from the SD sequence by a spacer region that is typically 9 nucleotides long in E.coli [26]. The 5” end of the coding sequence (~ 15 nucleotides) is also considered to be Withinthe 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 [30,3l]. This is thought to promote accessibility of the 305 subunit duringtranslation initiation [28,29,32,33].

DNA constructs relating to signal peptides are known from US836l744. HoWever, the 42DNA constructs disclosed in US836l744 differ significantly from the DNA constructs of thepresent invention both With respect to DNA sequence as Well as the performance of the signalpeptides. Moreover, the DNA constructs of US836l744 have not been synthetically evolvedand therefore do not eXhibit the technical effects of the DNA constructs disclosed in the present invention.

OBJ ECT OF INVENTIONThe 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. down- regulate) signal peptide performance.

A further object of the invention is to provide a simple and ineXpensive system of DNAconstructs, 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 recombinanteXpression plasmid typically used to produce secreted proteins. It is in the art a commonpractice to place the coding sequence of the signal peptide doWnstream of the vector encoded5 °UTR. Hence, the resulting TIR is a fusion of the 5 ”UTR and the first 5 codons of the signalpeptide in the TIR. The inventors hypothesized that such a TIR Would not function optimallyas it had not co-evolved With the ribosome. To test this hypothesis, as described in detail inthe DETAILED DESCRIPTION of the present specification, the inventors syntheticallyevolved the TIR in the presence of host cell ribosomes. The experimental results discussed inthe 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 PelBSP, Which Was initially the Worst performing signal peptide for production of ß-lactamase,but the best perforrning following synthetic evolution of the TIR. Thus, in summary, theperformance 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 systemcomprising: - DNA constructs, - expression vectors, - host cells, and - methods of production,for increasing the production yields of single chain antibody fragments, horrnones 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 signalpeptide performance, Wherein said DNA construct comprises: a. a Shine-Dalgarno 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 forexpressing 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 (periplasrr1ic 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-Dalgarno 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-Dalgarno 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 MalEsignal 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 aMalE 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 OmpAsignal 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 anOmpA 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 signalpeptide 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 PhoAsignal 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 DsbAsignal 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 DsbAsignal 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 PelBsignal 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 PelBsignal 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 36eXpresses 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 39eXpresses 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 asignal peptide of a sequence of one of SEQ ID 31; - said DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44eXpresses 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 eXpressesa 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 signalpeptide performance, Wherein said DNA construct comprises a sequence of one of SEQ ID -28.

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

In a preferred embodiment, said signal peptide encoding sequence comprises a sequence foreXpressing 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 (periplasn1ic 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 aMalE 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 anOmpA 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 PhoAsignal 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 DsbAsignal 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 PelBsignal 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 36eXpresses 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 39eXpresses 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 asignal peptide of a sequence of one of SEQ ID 31; said DsbA signal peptide encoding sequence of one of SEQ ID 42, 43 and 44eXpresses 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 eXpressesa 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 signalpeptide performance, Wherein said DNA construct comprises a sequence of one of SEQ ID 49, 5l, 53, 55 and 57.

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

In a preferred embodiment, said signal peptide encoding sequence comprises a sequence foreXpressing 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).

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 peptideencoding sequence of SEQ ID 58; - a sequence of SEQ ID 5l comprises the first 24 nucleotides of an OmpA signalpeptide encoding sequence of SEQ ID 59; - a sequence of SEQ ID 53 comprises the first 24 nucleotides of a PhoA signal peptideencoding sequence of SEQ ID 60; - a sequence of SEQ ID 55 comprises the first 24 nucleotides of a DsbA signal peptideencoding sequence of SEQ ID 6l; and/or - a sequence of SEQ ID 57 comprises the first 24 nucleotides of a PelB signal peptideencoding 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 peptideof sequence of SEQ ID 29; - said OmpA signal peptide encoding sequence of SEQ ID 59 eXpresses a signal peptideof sequence of SEQ ID 30; - said PhoA signal peptide encoding sequence of SEQ ID 60 expresses a signal peptideof sequence of SEQ ID 3l; - said DsbA signal peptide encoding sequence of SEQ ID 61 expresses a signal peptideof sequence SEQ ID 32; and/or - said PelB signal peptide encoding sequence of SEQ ID 62 expresses a signal peptideof 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 constructaccording to the above disclosed first, second or third aspects of the invention, Wherein theexpression 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 expressionvector of the fourth aspect of the invention, Wherein said host cell is preferably a bacterialcell, more preferably said bacterial cell is E. coli and most preferably E. coli strain BL2 l (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 l. A comparison of commonly used signal peptides. (A) An overview of the expressioncassettes used in this experiment. The TIR region represented by the boxed area, from the Shine-Dalgarno to the fifth codon of the signal sequence. The coding sequences for five commonly used signal peptides (MalESP, OmpASP, PhoASP, DsbASP, PelBSP) were cloned intothe pET28a vector, upstream of the mature coding sequences for ß-lactamase, scFvHERz orFtYfgMÅlS-lm. Protein production was induced for two hours, then a volume of cellscorresponding to 0.2 OD600 units of cells were harvested, separated by a l2 % SDS-PAGE andprotein levels deterrnined by immuno-blotting with antisera to ß-lactamase (B), or the poly-Histidine tag of scFvHERz (C) and FtYfgMÄlS-"O (D). To ensure that protein loading wasconsistent between the samples, the membranes were stained with Amido black after immuno-detection. “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) Asynthetic evolution approach was used to convert a TIRUNEVOLVED to a TIRSYNJEVOLVED. TheTIR is defined as the region from the Shine-Dalgamo (half-dome) to codon 5 of the signalpeptide. (B) mRNA has a high propensity to form structures, thus a TIRUNEVOLVED can besequestered into short- (top) or long-range structures (middle). Synthetic evolution shouldselect a TIRSYNJEVOLVED that is relaxed and more accessible to the ribosome (bottom). (C) An overview of the synthetic evolution process. A TIRLIBRARY was constructed by completelyrandomising the six nucleotides immediately upstream of the AUG start codon, and partiallyrandomising the six nucleotides immediately downstream of the AUG start codon (allowingsynonymous codons changes only). The TIRLIBRARY was transforrned into E. coli BL2l(DE3)pLysS and plated on increasing concentrations of ampicillin. A TIRSYNJEVOLVED was identifiedon the plate containing the highest concentration of ampicillin relative to the TIRUNEVOLVEDvariant. (D) ß-lactamase production levels from TIRUNEVOLVED / TIRSYNEVOLVED pairs wereassessed by immuno-blotting. ln this experiment, ß-lactamase production was induced for twohours, then a volume of cells corresponding to 0.2 OD600 units of cells were harvested, separatedby a l2 % SDS-PAGE and protein levels were determined by immuno-blotting with antisera toß-lactamase. To ensure that protein loading was consistent between the samples, the membranewas stained with Amido black after immuno-detection. “Pre° denotes the precursor form of theprotein, which contains the signal peptide fused version of ß-lactamase, which we presume tobe in the cytoplasm as the signal peptide is still present. “Mat° precursor denotes the matureversion of ß-lactamase, which presumably is in the periplasm as the signal peptide has been cleaved. (E) ß-lactamase activity from TIRUNEVOLVED / TIRSYNJEVOLVED pairs 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 ß-lactamase from either a TIRUNEVOLVED or a TIRSYNJEVOLVED. The diameter of the growth-inhibition zone was measured RS YNJEVOLVED for each experiment. In all cases, a TI conferred more resistant to ampicillin than TIRUNEVOLVED .

Figure 3. Time-course analysis of ß-lactamase production. (A) An illustration of theexperimental workflow used. (B) At each time point, a volume of cells was extracted, thenseparated by SDS-PAGE and immuno-blotted with antisera to ß-lactamase. Band intensitieswere obtained from immuno-blots by densitometric analysis and norrnalised to the highest- value.

Figure 4. A synthetically evolved TIR (TIRSYNEVOLVED) is transferable. (A) Expression levelsof ScFvHERz and FtYfgMÄlS-"O using five different signal peptides. In each instanceTIRUNEVOLVED / TIRSYNJEVOLVED pairs were assessed by immuno-blotting. The TIRSYN~EVOLVEDhad originally been selected for ß-lactamase (see Figure 2). In this experiment, proteinproduction was induced for two hours, then a volume of cells corresponding to 0.2 OD600 unitsof 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 aTIRSYNEVOLVED. (A) Production levels of hGH using five different signal peptides. In each instance the difference between TIRUNEVOLVED / TIRSYNJEVOLVED pairs were assessed byimmuno-blotting. The TIRSYNJEVOLVED had originally been selected for ß-lactamase (see Figure2). In this experiment, protein production was induced for two hours, then a volume of cellscorresponding to 0.2 OD600 units of cells were harvested, separated by a 12 % SDS-PAGE andprotein 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) Anoverview 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 underdenaturing- 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 constructWherein the DNA construct comprises: e. a Shine-Dalgarno sequence, f. an ATG start codon, g. a sequence of one of SEQ ID l-28 comprising said ATG start codon, and h. a signal peptide encoding sequence,Wherein the sequence of one of SEQ ID l-28 comprises at least the first 9 nucleotides of thesignal peptide encoding sequence. When the DNA construct comprise a sequence of one ofSEQ ID 15-28 then such a sequence comprises the Shine-Dalgamo sequence, said sequenceof one of SEQ ID l-l4 and at least the first 24 nucleotides of the signal peptide encoding SCqUCnCC .

The signal peptide encoding sequence may comprises a sequence for eXpressing a signalpeptide selected from MalE, OmpA, PhoA, DsbA and PelB. A specific signal peptideencoding 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 l.

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 peptidesequence, and/or (c) a signal peptide DNA sequence, are disclosed in Tables l and 2.

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

Signal Peptide Sequence Seq ID DNA sequence Seq IDpeptideMaIESP MKIKTGARILALSALTTMMFSA 29 ATGAAAATAAAAACAGG 58SALA TGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCCAC ATGAAAATTAAAACAGGT 34 GCACGCATCCTCGCATTA ATGTCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCCAC ATGAAGATCAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCCAC ATGAAAATAAAAACAGGTGCACGCATCCTCGCATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCCAC OrnpASP MKKTAUUAVALAGFATVAQA ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCCAC ATGAAGAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCCAC ATGAAGAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCCAC ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCCAC PhoASP MKQSTIALALLPLLFTPVTKA ATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCAC ATGAAGCAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCAC ATGAAGCAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCAC DsbASP MKKIWLALAGLVLAFSASA ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTT TTAGCGTTTAGCGCATCGGCGCAC ATGAAAAAGATTTGGCTG 42GCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGCAC ATGAAGAAAATTTGGCTG 43GCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGCAC ATGAAAAAGATTTGGCTG 44GCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGCAC Pe1B SP MKYLLPTAAAGLLLLAAQPAM 33 ATGAAATACCTGCTGCCG 62A ACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAG CCGGCGATGGCCCAC ATGAAGTATCTGCTGCCG 45ACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAC ATGAAATATCTGCTGCCG 46ACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAC ATGAAATATCTGCTGCCG 47ACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAC The invention further relates to an eXpression vector comprising the above-mentioned DNAconstruct. Additionally, the present invention relates to host cell comprising said eXpressionvector. Furthermore, the present invention relates to a recombinant protein expressed by saidhost 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 proteinshave been described in the EXAMPLES and EXPERIMENTAL PROCEDURES sections ofthis 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 peptideperformance of DNA constructs comprising a sequence of one of SEQ ID 1-28. HoWever, thepresent invention may alternatively be used for decreasing the signal peptide performance ofDNA 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 performanceby synthetically evolving the TIR. The present invention further provides a simple andineXpensive solution for increasing the production yields of secreted proteins in bacterial cellfactories. 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 TIRLIBRARIES. HoWever, in the presentinvention, said problem Was solved by using ß-lactamase protein, Which confers resistance toß-lactam antibiotics and can be easily screened; this embodiment of the present invention isdiscussed in detail in Examples 2 and 3. For proteins Where no simple screening assay isavailable it is possible to translationally-couple ß-lactamase to the recombinant protein andthereby solve potential problems. It is also possible to use the signal peptides in pET28a vectors from the present invention, Which possess a TIRSYNJEVOLVED and Which improvedproduction yields of a single chain antibody fragment, a hormone and another recombinantprotein in Escherichia coli; this embodiment of the invention is discussed in detail in Example A link between signal peptide performance and the efficiency of translation initiation hasbeen implied previously. Punginelli and co-Workers noted that non- synonymous nucleotidechanges in the signal peptide of the Tat-dependent formate dehydrogenase increasedproduction levels by up to 60-fold in E. coli [38]. And Ng and Sarkar noted that synonymouschanges to the Usp45sp signal peptide in Lactococcus lactis helped to increase productionlevels of a nuclease and an amylase by approximately 15% [39]. Both studies postulated thatthe nucleotide changes helped to relax mRNA structure that had sequestered the TIR.

The present invention also demonstrates that nucleotide changes in the TIR can influenceproduction 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 thatsignal peptides generally under-perforrn in protein production experiments because the TIR,encompassing the 5” UTR of the plasmid and the 5” terrninus 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 thismolecular explanation by demonstrating that a synthetic evolution process could improve theperformance of all commonly used signal peptides. As indicated earlier in the specification,the most striking example was PelBSP, which was initially the worst performing signal peptidefor production of ß-lactamase, but the best performing following synthetic evolution of theTIR as illustrated in Figure 2D. Thus, the performance of the signal peptide is largely coupled to the efficiency of translation initiation.

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

Example l - Production of periplasn1ic proteins with commonly used signal peptides Five signal peptides that are commonly used for the production of recombinant proteins in theperiplasm of E. coli were selected (MalESP, OmpASP, PhoASP, DsbASP and PelBSP; see Tablel; see SEQ-ID 29-33). The coding sequences for these signal peptides were cloned into thecommonly used pET28a expression plasmid, upstream of the coding sequence for ß-lactamase(Figure lA). To determine how efficiently the signal peptides supported the synthesis andsecretion of ß-lactamase the expression plasmids were transformed into the E. coli strainBL2l(DE3) pLysS and a mild induction protocol was used to initiate transcription (0.05 mMIPTG for 2 hours at 30 °C). Following the induction period, whole cells were collected, andproteins were separated by SDS-PAGE and immuno-blotted, so that the secreted (Mature) andnon-secreted (Precursor) ß-lactamase could be distinguished. The experiment indicated thatthere were large differences in production levels (Figure lB). MalESP, OmpASP, PhoASP,DsbASP supported a comparatively high-level of ß-lactamase production, whereas PelBSP did not. The experiment also indicated that there were significant differences in secretion efficiency between the different signal peptides. MalESP and PelBSP were effective insupporting the secretion of ß-lactamase to the periplasm, whereas OmpASP, PhoASP and DsbASP were deemed less effective as there was a prominent precursor band.

To evaluate the performance of the signal peptides with other recombinant proteins, they werefused to a single chain variable fragment that reco gnizes the human epidermal growth factorreceptor protein 2 protein (scFvHERz) and a soluble fragment of the periplasmic chaperoneYfgM from Francisella tularensis (FtYfgM45470). Again, there were considerable differencesin production levels across the different signal peptides (Figure lC and D). Moreover, therewere considerable differences between ß-lactamase, scFvHERz and FtYfgMÄlS-"O (Figure lB vsC vs D). Taken together, these observations demonstrate that signal peptide performance isvaried and unpredictable during the synthesis and secretion of recombinant periplasmicproteins. 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 plasn1ids used in the previous experiments had been assembled by geneticallysandwiching the nucleotide sequence encoding the signal peptide between the vector encoded5°UTR and the 5” end of the mature coding sequence for ß-lactamase, scFvHERz or FtYfgM45-170 (Figure 2A). Each expression plasmid therefore contained a different TIR (Table 2). Theinventors hypothesized that these TIRs might not be optimal for translation initiation as theyhad 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 TIRUNEVOLVED.

Synthetic (or directed) evolution was used to select TIRs that were more compatible with thehost cell ribosomes. ln the experiment, TIRLIBRARIEScontaining the MalESP, OmpASP, PhoASP, DsbASP and PelBSP fused to ß-lactamase. In the design of the TIRLIBRARIES, the six nucleotides immediately upstream from the AUG start were created from expression plasn1ids codon were completely randomized, and the six nucleotides immediately downstream fromthe AUG start codon were randomized with synonymous codon changes only (Figure 2C)[34,35]. Each TIRLIBRARY theoretically contained >l8,000 expression plasmids with adifferent TIR. The TIRLIBRARIES were transforrned into BL2l(DE3) pLysS and plated onto LBagar 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 syntheticallyevolved (TIRSYNJEVOLVED) (Table 2).

Table 2. Nucleotide sequences of the TIRUNEVOLVED and corresponding TIRSYNJEVOLVED used in this study. The TIR is defined as the region from the Shine-Dalgarno to codon five of the signal peptide.

Signal TIR Sequence Seqpeptide IDMalESP UNEVOLVED ÉÃTÃÉÄÉTIIÄTÉÉÄïÉåÃÅCCÉCÉIAGATATACCGATGAAAA 48/49 PhoASP UNEVOLVED ïïëåciflåäëaïfqèf/ÅGCÉIÄÉÉÉAGATATACCGATGAAAC 52/53 W 21 DsbASP UNEVOLVED ïïTfifšiêëëëTfifè/Éfè/ÉIÉÉÉAGATATACCGATGAAAA 54/55 PelBSP UNEVOLVED ïëïiëiêgréëë/ÉÉÉIÉÉÉÉAGATATACCGATGAAAT 56/57 lUnderlined region Was randomised during the synthetic evolution process 2Nucleotides marked in bold text Were changed in TIRSYNFEVOLVED 3SEQ ID is indicated for underlined region (referred to as “short sequence” in the sequence listing) andfull nucleotide sequence (referred to as “full sequence” in the sequence listing), respectively Expression plasmids containing either a TIRUNEVOLVED or TIRSYNJEVOLVED Were re-transformed into BL21(DE3) pLysS and the production levels of ß-lactamase compared byimmuno-blotting. After a two-hour induction period We observed that the production levels ofperiplasmic ß-lactamase Were significantly higher When using a TIRSYNJEVOLVED compared tothe TIRUNEVOLVED (Figure 2D). Note that production of ß-lactamase from each TIRUNEVOLVEDWas undetectable on these blots because the difference With the TIRSYN~EVOLVED Was too largeto capture at this time point (see below). Consistent With this observation, disc diffusionassays confirmed that the TIRSYNJEVOLVED supported a higher level of resistance to ampicillinthan the TIRUNEVOLVED (Figure 2E). Figures 2E and 2D illustrate comparative experimentsusing TIRUNEVOLVED and TIRSYNJEVOLVED Wherein (i) TIRSYNJEVOLVED comprised SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRUNEVOLVED comprised SEQ ID of 48, 50, 52, 54 and 56,respectively.

The inventors speculate that the difference in production levels from the TIRUNEVOLVED/TIRSYNJEVOLVED pairs Was a result of mRNA relaxation, but the inventors Were unable tosupport this speculation by using mRNA fold prediction programs. The lack of a correlationcould reflect the fact that (1) mRNA relaxation is not the sole determinant, (2) mRNAstructure is notoriously difficult to predict, and/or (2) existing algorithms only handle shortstretches of nucleotides (not an entire mRNA). Nevertheless, the experiment doesdemonstrate that all signal peptides Were under-performing When a TIRUNEVOLVED Was used.And significantly, the performance of all signal peptides could be improved by synthetically evolving the TIRUNEVOLVED. This phenomenon Was most easily seen With PelBSP, Which gave the loWest levels of ß-lactamase production When expressed from a TIRUNEVOLVED (Figure IB) but the highest When expressed from a TIRSYNJEVOLVED (Figure 2E). Synthetic evolution of theTIR had therefore “converted° the PelBSP from a “poor-performing” signal peptide to a “top-performing” signal peptide without changing a single amino acid. The data thereforedemonstrate that signal peptide performance is tightly coupled to translation initiation in bacterial cell factories.

Example 3 - Production of recombinant periplasmic proteins using a TIRSYNJEVOLVED In the previous series of experiments a mild induction protocol had been used (0.05 mM IPTGfor 2 hours at 30 °C), so that differences in protein production could be assessed in theabsence of a metabolic load on the cell. The concern about metabolic load largely relates tothe Sec translocon, which is believed to be a bottleneck in the production of periplasmicproteins [36,37]. When production levels of periplasmic proteins are too high, the transloconcould become saturated and the recombinant protein may be retained in the cytoplasm. Todetermine if eXpression plasmids with a TIRSYNEVOLVED would saturate the Sec translocon,the inventors induced with either a low (0.05 mM) or a high (0.5 mM) IPTG concentrationand monitored production over a 5-hour period (Figure 3A). It was observed that, at all butone time-point, a TIRSYNJEVOLVED produced more periplasmic ß-lactamase than thecorresponding TIRUNEVOLVED (Figure 3B). This observation was made at both low and highconcentrations of IPTG. These time-course eXperiments therefore indicated that the Sectranslocon was able to cope with the increased production levels that were reached using aTIRSYNJEVOLVED. Figures 3B illustrates comparative experiment using TIRUNEVOLVED andTIRSYNJEVOLVED wherein (i) TIRSYNJEVOLVED comprised SEQ ID l5, 18, 2l, 23 and 26, and (ii)TIRUNEVOLVED comprised SEQ ID of 48, 50, 52, 54 and 56, respectively.

RS YNJEVOLVED Example 4 - Using TI as a generic solution M45-l70 In this set of experiments the coding sequences of scFvHER2 and FtYfg were RUNEVOLVED expressed as fusions to the original five signal peptides, using both the TI and TIRSYNFEVOLVED pairs. The eXpression plasmids were again transforrned into BL2l(DE3)pLysS and production was monitored using a mild induction protocol (005 mM IPTG for 2hours at 30 °C). As we had observed for ß-lactamase, the TIRSYNJEVOLVED always produced more protein than the corresponding TIRUNEVOLVED (Figure 4). It was noted that signal peptide performance was varied; the most effective signal peptide for production of scFvHERz was PhoASP, whilst the most effective for FtYfgMÄlS-”O was MalESP. Thus, signal peptideperformance might partly be explained by compatibility of the signal peptide. Figure 4 illustrates comparative eXperiments using TIRUNEVOLVED and TIRSYNJEVOLVED wherein (i)TIRSYNJWOLVED comprised SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRUNEVOLVED comprised SEQ ID of 48, 50, 52, 54 and 56, respectively.

A similar approach was taken to produce the human growth hormone (hGH). Here weobserved that the most effective TIRSYN~EVOLVED for production of hGH was the one coupledto the PelBSP (Figure 5A). To assess how much more protein was produced the N-terrninallyHis-tagged hGH was purified by Immobilized Metal Affinity Chromatography (IMAC), theHis-tag removed by proteolytic processing, and the sample polished by Size EXclusionChromatography (SEC) (Figure 5B). The yield of purified hGH was more than 3-fold higherusing the TIRSYNJEVOLVED compared to the TIRUNEVOLVED (2.56 mg/L vs 0.79 mg/L).Importantly, we could not detect any difference in the quality of the purified hGH, as judgedby monodispersity of the sample following SEC (Figure 5C), the proportion of protein thathad formed disulphide bonds (Figure 5D), or the activity of the protein when tested by theMTS cell proliferation assay (Figure 5E). Figures 5A, 5C, 5D and 5E illustrate comparativeRUNEvoLvED and TIRsYNjvoLvED Wherein (i) TIRSYNJEVOLVED eXperiments using TI SEQ ID 15, 18, 21, 23 and 26, and (ii) TIRUNEVOLVED comprised SEQ ID of 48, 50, 52, 54 and comprised 56, respectively.

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

EXPERIMENTAL PROCEDURESMolecular cloning The sequences encoding MalESP, OmpASP, PhoASP, DsbASP, PelBSP, ß-lactamase, hGH andFtYfgMÅlS-”O were chen1ically synthesised (Genscript, USA). The sequence encoding scFvHERzwas 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 Ndelrestriction 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 approximately45 nucleotides in length and was partly degenerate. The design enabled completerandomization of the six nucleotides upstream of the AUG start codon, and partialrandomization 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 ”). Itoverlapped with the forward primer by l3 nucleotides thus allowing circularization of thePCR product by homologous recombination in E. coli MCl06l. The PCR was carried outusing the Q5 polymerase (New England Biolabs, UK) in a program that consisted of 94 °C for5 min and then 30 cycles of 95 °C for 45 s, 48-68 °C for 45 s (using a gradient therrnocycler),72 °C for 6 min and a final elongation step of 72 °C for 5 min. Specific PCR products thatwere amplified at the lowest annealing temperature were treated with Dpnl, then transformedinto chemically competent E. coli MCl06l. The transformation was seeded into 100 mL ofLuria-Bertani containing 50 ug/mL kanamycin and incubated overnight at 37 °C. lsolation ofthe TIRLIBRARIES was carried out using ten E.N.Z.A DNA mini kit purification columns (Omega Biotek, USA) and pooling of the eluates.

TIRLIBRARIES were screened by transforming chemically competent BL2l (DE3) pLysS and identifying clones that survived on the highest concentration of ampicillin. Here 0.5 ug of theTIRLIBRARY was transforrned into 50 uL of chemically competent BL2l(DE3) pLysS usingstandard protocols. The entire transformation was then seeded into 3 mL of LB containing 50ug/mL kanamycin and 34 ug/mL chloramphenicol. Cultures were grown at 37 °C withshaking for l6 h. Cultures were then back-diluted (l:50) into 5 mL of LB containing 50ug/mL kanamycin and 34 ug/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-ß-Dthiogalactopyranoside (IPTG) and increasing concentrations of ampicillin (100-5000 ug/mL).

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

Immuno-blotting Cultures Were groWn at 37 °C With shaking for 16 h, then back-diluted (l:50) into 5 mL of LBcontaining 50 ug/mL kanamycin and 34 ug/mL chloramphenicol and incubated as before untilan OD600 of ~0.3-0.5 Was reached. EXpression of the coding sequence Was induced With 0.05mM IPTG for 2 h at 30 °C. A Volume of cells corresponding to an OD600 of either 0.02 or 0.2Was harvested by centrifugation then resuspended in 2X Laemlli loading buffer [l25 mM Tris-HCl pH 6.8, 4% SDS, 3% Glycerol, 0.02% bromophenol blue, 20% ß-mercaptoethanol].Proteins Were separated by 12% SDS-PAGE then transferred to a nitrocellulose membraneusing a sen1i-dry transfer apparatus (Bio-Rad, USA). The nitrocellulose membranes Wereprobed With an antibody against either ß-lactamase (Therrno Scientific, USA) or the poly-histidine tag (His-Probe, ThermoFisher Scientific, USA). Binding Was detected using anti-mouse IgG linked to horseradish peroXidase (GE healthcare, USA) and a SuperSignal Westfemto 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 ug/mL kanamycin and 34 ug/mL chloramphenicoluntil an OD600 of ~0.3. A Volume of cells corresponding to an OD600 of 0.002 Was then platedonto LB agar (lacking all antibiotics). A sterile filter disc containing 2 mg ampicillin Was thenplaced on top of the cells and the plates Were incubated at 37 °C for l6 h. Zones of growth inhibition Were measured using a standard ruler.

Purification of hGH Expression plasn1ids harboring pET28a pelB-hGH Were transforrned into the eXpression hostBL2l(DE3) pLysS and groWn on LB agar plates containing 50 ug/mL kanamycin and 34 ug/mL chloramphenicol. Single colonies Were used to inoculate 100 mL of LB plus antibiotics medium Which Was groWn ovemight at 37 °C With shaking at 180 RPM. Overnightpre-cultures Were used to inoculate 2 L flasks containing 1 L of LB media plus antibiotics, toa starting OD600 of 0.05. Cultures Were groWn to an OD600 of 0.7, at Which point, flasks Wereincubated on ice for 10 minutes. Induction proceeded With the addition of 0.01 mM IPTG andincubation for 16 hours at 18 °C With shaking at 180 RPM. Cells Were harvested for 20minutes at 4,000 X g. Cell pellets Were resuspended in 50 mL suspension buffer (50 mM TrispH 8.0, 500 mM NaC1, 20 mM imidazole pH 8.0 and lx protease inhibitor cocktail(cOmp1ete, Roche, USA)). Cell suspensions Were homogenized With a glass Douncehomogenizer followed by cell disruption using an Avestin emulsiflex C3 high-pressurehomogenizer (Avestin, Canada). Cell debris Was removed by centrifugation at 20,000 X g for30 minutes. Samples Were applied to 2.5 mL Ni-sepharose (GE Healthcare) and batchincubated at 4 °C for one hour on a benchtop roller. The column Was Washed With 20 columnvolumes (50 mL) of Wash buffer (50 mM Tris pH 8.0, 500 mM NaC1 and 50 mM imidazolepH 8.0), followed by elution With 30 mL of elution buffer (50 mM Tris pH 8.0, 500 mM NaC1and 500 mM imidazole pH 8.0). The elution fraction Was concentrated and buffer eXchanged(50 mM Tris pH 8.0, 150 mM NaC1 and 20 mM imidazole) using a centrifugal filter With anorr1inal MWCO of 10 kDa (Amicon, Merck Millipore). The N-terrninal his-tag Wasproteolytically removed With TEV protease (purified in-house) at a 1:10 Weight ratio andallowed to incubate overnight at 4 °C. Samples Were reverse Ni purified, concentrated andapplied to size eXclusion chromatography using a SuperdeX 200 10/300 GL column (GEHealthcare, Sweden) in 50 mM Tris pH 8.0 and 100 mM NaC1. Relevant fractions Werepooled, 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 containing10% FBS, 2mM glutarnine and 1% penicillin streptomycin (Gibco/Therrno Fisher Scientific)at 37 °C in a humidified atmosphere at 5% C02. Cell proliferation following titration ofpurified hGH Was determined according to the CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay (MTS) protocol (Promega). Briefly, 1X104 MCF7 cells Were seeded in triplicate, in 100 uL 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 mediumat a final concentration ranging from 0 to 400 ng/mL. Cell proliferation Was assessed after 48hours 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 (7)

1. DNA construct suitable for regulating signal peptide performance, Wherein said DNAconstruct comprises a sequence of one of SEQ ID 49, 51, 53, 55 and 57.

2. DNA construct according to claim 1, Wherein said DNA construct also comprises a signal peptide encoding sequence.

3. DNA construct according to claim 2, Wherein the signal peptide encoding sequencecomprises a sequence for eXpressing a signal peptide selected from MalE, OmpA, PhoA, DsbA and Pelb.

4. DNA construct according to any one of the previous claims 2-3, Wherein said signal peptide encoding sequence is a sequence of one of SEQ ID 58-62.

5. DNA construct according to any one of the previous claims 2-4, Wherein said signalpeptide encoding sequence eXpresses a signal peptide of a sequence of one of SEQ ID29-33.

6. DNA construct according to claim 4, Wherein: 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 asignal peptide of sequence of SEQ ID 30; - said PhoA signal peptide encoding sequence of SEQ ID 60 eXpresses asignal peptide of sequence of SEQ ID 31; - said DsbA signal peptide encoding sequence of SEQ ID 61 eXpresses asignal 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 33.

7. DNA construct according to any one of the previous claims, Wherein said DNA construct further comprises a recombinant protein encoding sequence. EXpression vector comprising the DNA construct according to any one of c1aims 1-7,Wherein the eXpression Vector is preferab1y a p1asmid, more preferab1y PET eXpression vector, and most preferab1y pet28A Host ce11 comprising the eXpression Vector according to c1aim 8, Wherein said host ce11is preferab1y a bacteria1 ce11, more preferab1y said bacteria1 ce11 is E. co1i and most preferab1y E. co1i strain BL21(DE3) pLysS. Method of eXpressing a recombinant protein, comprising the steps of:a. introducing a DNA construct according to c1aim 7 into an eXpression vector;b. introducing the eXpression vector into a host ce11;c. growing the host ce11; and d. recovering the recombinant protein from the host ce11. RNA mo1ecu1e expressed by a DNA construct according to any one of the c1aims 1-7. Use of a DNA construct according to any one of the c1aims 1-7 for regu1ating signa1 peptide performance. Use of a DNA construct according to c1aim 12 for regu1ating signa1 peptideperformance, Wherein said regu1ating signa1 peptide performance is down-regu1ating signa1 peptide performance.