Method for preparing recombinant protein by reducing rnpA gene expression

Abstract

The present invention relates to a method for improving the yield of a difficult-to-express recombinant protein in a recombinant microorganism, and more particularly to a method for improving the yield of a difficult-to-express recombinant protein by using a recombinant microorganism into which a gene encoding a target protein and an sRNA against a gene encoding ribonuclease P are introduced. According to the present invention, the expression of large recombinant proteins, difficult-to-express proteins and useful proteins can be significantly increased by reducing the expression of the rnpA gene.

Description

Technical Field

[0001] The present invention relates to a method for improving the yield of a recombinant protein in a recombinant microorganism, and more particularly to a method for improving the yield of a recombinant protein by using a recombinant microorganism into which a gene encoding a target protein and an sRNA targeting a gene encoding ribonuclease P are introduced. Background Art

[0002] The development of gene manipulation technology has led to many studies focusing on the use of bacteria and a variety of plants and animals to produce large amounts of useful proteins. Existing host cells for producing large amounts of useful proteins include bacteria such as Escherichia coli (E. coli) and yeasts such as Pichia pastoris (P. pastoris). Among these host cells, Escherichia coli is the most widely used and has been the most studied (Choi et al., Chem. Eng. Sci., 66: 876, 2006; Lee, Trends Biotechnol., 14: 98, 1996).

[0003] However, if the useful protein to be prepared is larger than the naturally occurring protein or difficult to express, many problems will arise. If the size of a protein is large, due to the lack of messenger RNA (mRNA), the translation of the protein is difficult, and thus the expression of the protein of the required length is difficult. In addition, due to proteolysis and RNAse-induced mRNA degradation, recombinant proteins that do not naturally exist in Escherichia coli are difficult to express (GoBringer et al., J Bacteriol., 188: 6816, 2006; Olson et al., PLoS Pathog., 7 (2): e1001287, 2011; Jung et al., Biochem Biophys Res Commun., 186 (3): 1463, 1992; Altman et al., Phil Trans R Soc., 366, 2011; Turrini et al., PLos One., 7 (3): e32456, 2012).

[0004] Therefore, the inventors of the present invention have made extensive efforts to develop a protein expression system for increasing the yield of difficult-to-express foreign proteins, and as a result, found that by reducing the expression of the rnpA gene (which is a component of ribonuclease P) during the expression of foreign proteins by introducing genes encoding foreign proteins, the expression of difficult-to-express foreign proteins is increased, thereby achieving the present invention. Summary of the invention

[0005] Technical issues

[0006] A main object of the present invention is to provide a recombinant microorganism, into which a gene encoding a target protein and an sRNA targeting a gene encoding ribonuclease P are introduced to increase the yield of a foreign protein that is difficult to express.

[0007] Another object of the present invention is to provide a method for preparing a target protein by culturing the above recombinant microorganism.

[0008] Technical Solution

[0009] In order to achieve the above object, the present invention provides a recombinant vector for expressing a target protein and a recombinant microorganism, wherein the recombinant vector comprises a gene encoding the target protein and an sRNA targeting a gene encoding ribonuclease P, and the recombinant vector is introduced into the recombinant microorganism.

[0010] The present invention also provides a recombinant microorganism, into which a gene encoding a target protein and an sRNA targeting a gene encoding ribonuclease P are introduced.

[0011] The present invention also provides a method for preparing a target protein, comprising the following steps: (a) preparing the target protein by culturing the above-mentioned recombinant microorganism and inducing the expression of the target protein in the recombinant microorganism; and (b) recovering the prepared target protein.

[0012] The present invention also provides a method for preparing a target protein, comprising the following steps: expressing and preparing the target protein by culturing a recombinant microorganism into which a gene encoding the target protein is introduced; recovering the prepared target protein, wherein the expression of ribonuclease P is reduced to increase the expression of the target protein. Sequence Listing <110> KAIST <120> Method for preparing recombinant protein by reducing rnpA gene expression <130> PF-B1895-CN <140> PCT / KR2015 / 013419 <141> 2015-12-09 <150> 10-2014-0175780 <151> 2014-12-09 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> rnpA sRNA <400> 1 gtggttaagc tcgcatttcc cagg 24 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> rnpA sRNA <400> 2 gtggttaagc tcgcatttcc 20 <210> 3 <211> 16 <212> DNA <213> Artificial sequence <220> <223> rnpA sRNA <400> 3 gtggttaagc tcgcat 16 <210> 4 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 cctgggaaat gcgagcttaa ccactttctg ttgggccatt gcattg 46 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 5 gcaaccatta tcaccgcca 19 <210> 6 <211> 1721 <212> DNA <213> Artificial sequence <220> <223> f <400> 6 atttcaaaa aagagtgagt gacatggaac caaaaacaaa aaaacagcgt tcgctttata tcccttacgc tggccctgta ctgctggaat ttccgttgtt gaataaaggc agtgccttca 120 gcatggaaga acgccgtaac ttcaacctgc tggggttact gccggaagtg gtcgaaacca tcgaagaaca agcggaacga gcatggatcc agtatcaggg attcaaaacc gaatcgaca aacacatcta cctgcgtaac atccaggaca ctaacgaaac cctcttctac cgtctggtaa acaatcatct tgatgagatg atgcctgtta tttatacccc aaccgtcggc gcagcctgtg 360 agcgtttttc tgagatctac cgccgttcac gcggcgtgtt tatctcttac cagaaccggc 420 acaatatgga cgatattctg caaaacgtgc cgaaccataa tattaaagtg attgtggtga ctgacggtga acgcattctg gggcttggtg accagggcat cggcgggatg ggcattccga 540 tcggtaaact gtcgctctat accgcctgtg gcggcatcag cccggcgtat acccttccgg 600 tggtgctgga tgtcggaacg grandmother agctgcttaa cgatccgctg fathergggct ggcgtaatcc gcgtatcact gacgacgaat actatgaatt cgttgatgaa tttatccagg ctgtgaaaca acgctggcca gacgtgctgt tgcagtttga agactttgct caaaaaaatg 780 cgatgccgtt acttaaccgc tatcgcaatg aaatttgttc ttttaacgat gacattcagg 840 gcactgcggc ggtaacagtc ggcacactga tcgcagcaag ccgcgcggca ggtggtcagt 900 taagcgagaa aaaaatcgtc ttccttggcg caggttcagc gggatgcggc attgccgaaa 960 tgatcatctc ccagacccag cgcgaaggat taagcgagga agcggcgcgg cagaaagtct 1020 ttatggtcga tcgctttggc ttgctgactg acaagatgcc gaacctgctg cctttccaga 1080 ccaaactggt gcagaagcgc gaaaacctca gtgactggga taccgacagc gatgtgctgt 1140 cactgctgga tgtggtgcgc aatgtaaaac cagatattct gattggcgtc tcaggacaga 1200 ccgggctgtt tacggaagag atcatccgtg agatgcataa acactgtccg cgtccgatcg 1260 tgatgccgct gtctaacccg acgtcacgcg tggaagccac accgcaggac attatcgcct 1320 ggaccgaagg taacgcgctg gtcgccacgg gcagcccgtt taatccagtg gtatggaaag 1380 ataaaatcta ccctatcgcc cagtgtaaca acgcctttat tttcccgggc atcggcctgg 1440 gtgttattgc ttccggcgcg tcacgtatca ccgatgagat gctgatgtcg gcaagtgaaa 1500 cgctggcgca gtattcacca ttggtgctga acggcgaagg tatggtactg ccggaactga 1560 aagatattca gaaagtctcc cgcgcaattg cgtttgcggt tggcaaaatg gcgcagcagc 1620 aaggcgtggc ggtgaaaacc tctgccgaag ccctgcaaca ggccattgac gataatttct 1680 ggcaagccga ataccgcgac taccgccgta cctccatcta a 1721 <210> 7 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> srtA <400> 7 atgaaaaaat ggaccaaccg cctgatgacc attgcgggcg tggtgctgat tctggtggcg 60 gcgtatctgt ttgcgaaacc gcatattgat aactatctgc atgataaaga taaagatgaa 120 aaaattgaac agtatgataa aaacgtgaaa gaacaggcga gcaaagataa aaaacagcag 180 gcgaaaccgc agattccgaa agataaaagc aaagtggcgg gctatattga aattccggat 240 gcggatatta aagaaccggt gtatccgggc ccggcgaccc cggaacagct gaaccgcggc 300 gtgagctttg cggaagaaaa cgaaagcctg gatgatcaga acattagcat tgcgggccat 360 acctttattg atcgcccgaa ctatcagttt accaacctga aagcggcgaa aaaaggcagc 420 atggtgtatt ttaaagtggg caacgaaacc cgcaaatata aaatgaccag cattcgcgat 480 gtgaaaccga ccgatgtggg cgtgctggat gaacagaaag gcaaagataa acagctgacc 540 ctgattacct gcgatgatta taacgaaaaa accggcgtgt gggaaaaacg caaaattttt 600 gtggcgaccg aagtgaaa 618 <210> 8 <211> 1290 <212> DNA <213> Artificial Sequence <220> <223> Cat2 <400> 8 atggagtggg aagagatata taaagagaaa ctggtaactg cagaaaaagc tgtttcaaaa 60 atagaaaacc atagcagggt agtttttgca catgcagtag gagaacccgt agatttagta 120 aatgcactag ttaaaaataa ggataattat ataggactag aaatagttca catggtagct 180 atgggcaaag gtgaatatac aaaagagggt atgcaaagac attttagaca taatgcttta 240 tttgtaggcg gatgtactag agatgcagta aattcaggaa gagcagatta tacaccttgt 300 ttttctatg aagtgccaag tttgtttaaa gaaaaacgtt tgcctgtaga tgtagcactt 360 attcaggtaa gtgagccaga taaatatggc tactgcagtt tggagttttc caatgactat 420 accaagccag cagcagaaag tgctaagctt gtattgcag aagtgaataa aaacatgcca 480 agaactcttg gagattcttt tatacatgta tcagatattg attatatagt ggaagcttca 540 cacccattgt tagattgca gcctcctaaa ttgggagatg tagaaaagc cataggagaa 600 aactgtgcat ctttaattga agatggagct actctcagc ttggaatagg tgctatacca 660 gatgcggtac ttttattct aaagaacaa aagaatttag gatacattc tgagatgata 720 tcagatggtg tgatggact ggtgaggca ggggttatca atacaagaa aagaccctc 780 catccaggca aaatagttgt aacatttta atgggaaca aaaaattata tgatttgta 840 aacaataatc caatggtaga aacttattct gtagatttag taaataatcc actggtaatt 900 atgaaaaatg acaatggt ttcaataat tcttgtgttc aagtagactt aatgggacaa 960 gtatgttctg aaagtatagg attgaacag atagtggag tgggaggcca ggtagatttt 1020 attagaggag ctaatctatc aaagggtgga aaggctatta tagctatacc ttccacagct 1080 ggaaaaggaa aagtttcaag aataactcca cttctagata ctggtgctgc agttacaact 1140 tctagaaatg aagtagatta tgtagttact gaatatggtg ttgctcatct taagggcaaa 1200 actttaagaa atagggcaag agctctaata aatatcgctc atccaaaatt cagagaatca 1260 ttaatgaatg aatttaaaaa gagattttag 1290 <210> 9 <211> 1518 <212> DNA <213> Artificial Sequence <220> <223> CYP73A5 <400> 9 atggacctcc tcttgctgga gaagtcttta atcgccgtct tcgtggcggt gattctcgcc 60 acggtgattt caaagctccg cggcaagaaa ttgaagctac ctccaggtcc tataccaatt 120 ccgatcttcg gaaactggct tcaagtcgga gatgatctca accaccgtaa tctcgtcgat 180 tacgctaaga aattcggcga tctcttcctc ctccgtatgg gtcagcgaaa cctagtcgtc 240 gtctcctcac cggatctaac aaaggaagtg ctcctcactc aaggcgttga gtttggatcc 300 agaacgagaa acgtcgtgtt cgacattttc accgggaaag gtcaagatat ggtgttcact 360 gtttacggcg agcattggag gaagatgaga agaatcatga cggttccttt cttcaccaac 420 aaagttgttc aacagaatcg tgaaggttgg gagtttgaag cagctagtgt tgttgaagat 480 gttaagaaga atccagattc tgctacgaaa ggaatcgtgt tgaggaaacg tttgcaattg 540 atgatgtata acaatatgtt ccgtatcatg ttcgatagaa gatttgagag tgaggatgat 600 cctcttttcc ttaggcttaa ggctttgaat ggtgagagaa gtcgattagc tcagagcttt 660 gagtataact atggagattt cattcctatc cttagaccat tcctcagagg ctatttgaag 720 atttgtcaag atgtgaaaga tcgaagaatc gctcttttca agaagtactt tgttgatgag 780 aggaagcaaa ttgcgagttc taagcctaca ggtagtgaag gattgaaatg tgccattgat 840 cacatccttg aagctgagca gaagggagaa atcaacgagg acaatgttct ttacatcgtc 900 gagaacatca atgtcgccgc gattgagaca acattgtggt ctatcgagtg gggaattgca 960 gagctagtga accatcctga aatccagagt aagctaagga acgaactcga cacagttctt 1020 ggaccgggtg tgcaagtcac cgagcctgat cttcacaaac ttccatacct tcaagctgtg 1080 gttaaggaga ctcttcgtct gagaatggcg attcctctcc tcgtgcctca catgaacctc 1140 catgatgcga agctcgctgg ctacgatatc ccagcagaaa gcaaaatcct tgttaatgct 1200 tggtggctag caaacaaccc caacagctgg aagaagcctg aagagtttag accagagagg 1260 ttctttgaag aagaatcgca cgtggaagct aacggtaatg acttcaggta tgtgccattt 1320 ggtgttggac gtcgaagctg tcccgggatt atattggcat tgcctatttt ggggatcacc 1380 attggtagga tggtccagaa cttcgagctt cttcctcctc caggacagtc taaagtggat 1440 actagtgaga aaggtggaca attcagcttg cacatcctta accactccat aatcgttatg 1500 aaaccaagga actgttaa 1518 <210> 10 <211> 1527 <212> DNA <213> Artificial Sequence <220> <223> CYP98A3 <400> 10 atgtcgtggt ttctaatagc ggtggcgaca atcgccgccg tcgtatccta caagctaatc 60 120. caacggctaa gatacaagtt cccaccaggc ccaagcccca agccgatcgt cggtaacctc 180. tacgacataa aaccggtccg gttcagatgt tactacgagt gggctcaatc ttatggacca atcatatcgg tctggatcgg ttcaattcta aacgtggtcg tatctagcgc cgagctagca 240 aaagaagttc tgaaagaaca cgaccagaa ctcgccgacc ggcaccgga cagatcgacg gaagcattta gccgcaacgg tcaggatctt atatgggccg atatgggcc tcattacgtg 360 aaggtgagaa aagtttgcac gcttgagctc ttcacaccga aacgactcga gtctctcaga cctatccgtg aagatgaagt caccgccatg gttgaatccg tcttcagaga ctgtaacctt cctgaaaaca gagcaaaagg tttacaactg aggaagtact taggagcggt tgcgttcaac aacataacgc ggctagcctt tgggaagcgt tttatgaacg ctgaaggtgt tgtggacgag caagggcttg agttcaaggc catagtatcc aacggtctga agctaggtgc ttcactgtca 660. atagctgaac acatcccgtg gctcaggtgg atgtttccgg ctgatgagaa ggcgtttgct gagcacgggg ctcgtcgtga ccgcctcact cgagctatca tggaggagca tactttggcc 780 cgtcaaaagt ctagtggagc gaaacagcat ttcgttgatg cgttgctaac gttgaaggat 840 cagtatgatc ttagtgagga tactatcatt ggtcttctat gggatatgat cacggcaggg 900 atggacacga cagcgataac agcggaatgg gcgatggcgg aaatgatcaa gaatccaaga 960 gtgcaacaaa aagtgcaaga agagttcgac agagtggttg gacttgaccg gatcttaacc 1020 gaggcagatt tctcccgctt accttacttg caatgcgtgg tgaaagagtc attcaggctg 1080 catcctccaa cgcctctaat gctacctcac cgaagcaacg cagatgtcaa gatcggaggc 1140 tatgatattc ccaaaggatc aaacgttcat gtgaatgtgt gggctgtggc tagagacccg 1200 gctgtatgga aaaatccatt tgagtttaga ccagagagat tcttggaaga agatgttgac 1260 atgaagggtc atgattttag gctgcttccg tttggagctg gaagacgggt ttgtcccggt 1320 gcacaacttg gtatcaattt ggtaacttcg atgatgagtc atttgcttca ccattttgtt 1380 tggacacctc ctcaagggac taaaccggag gagattgaca tgtctgaaaa ccctggactc 1440 gttacttaca tgcgtacccc tgtgcaagcg gttgcaacgc ctcggttgcc ttcggatctg 1500 tacaaacgcg tgccttacga tatgtaa 1527 <210> 11 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 11 ccatggatat tcaaaaaaga gtgagt 26 <210> 12 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 12 tctagattag atggaggtac ggcggta 27 <210> 13 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 13 gaattcatgg agtgggaagagatatata 28 <210> 14 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 14 ggtaccctaa aatctctttt taaattcatt 30 <210> 15 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 15 gcgaagctta cagttccttg gtttcataa 29 <210> 16 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 16 gtacatatga tggacctcctcttgctgg 28 <210> 17 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 ggatccatgt cgtggtttct aatagc 26 <210> 18 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 gaattcttac atatcgtaag gcacgc 26 BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the gene map of plasmid pTetly2glyAN-rnpA(sRNA).

[0014] Figure 2 The results of SDS-PAGE analysis of the expression levels of silk proteins composed of 16 repeat sequences and silk proteins composed of 32 repeat sequences are shown. (Lane 1: markers showing the standard molecular weight of proteins; Lanes 2 and 3: at an OD of 0.4 600 The results of protein expression induction in strains transformed with plasmid pSH16; Lanes 4 and 5: at OD 0.4 600 The results of protein expression induction in strains transformed with plasmids pSH16 and pACYC184-rnpA (sRNA); Lanes 6 and 7: at OD 0.4 600 The results of protein expression induction in strains transformed with plasmid pSH32; Lanes 8 and 9: at OD 0.4 600Results of inducing protein expression in strains transformed with plasmids pSH32 and pACYC184-rnpA(sRNA).

[0015] Figure 3 The results of SDS-PAGE analysis of the expression level of silk protein composed of 64 repeat sequences are shown. (Lane 1: marker showing the standard molecular weight of the protein; Lane 2: at an OD of 0.4 600 The results of protein expression induction in the strain transformed with plasmid pSH64; Lane 3: at OD 0.4 600 Results of protein expression induction in strains transformed with plasmids pSH64 and pACYC184-rnpA(sRNA).

[0016] Figure 4a The results of SDS-PAGE analysis of the expression level of silk protein composed of 96 repeat sequences are shown. (Lane 1: marker showing the standard molecular weight of the protein; Lane 2: at an OD of 0.4 600 The results of protein expression induction in strains transformed with plasmid pSH96; Lane 3: at OD 0.4 600 Results of protein expression induction in strains transformed with plasmids pSH96 and pACYC184-rnpA(sRNA).

[0017] Figure 4b The results obtained by inducing protein expression in strains transformed with plasmid pSH96 and plasmid pSH96 plus pTetgly2glyAN-rnpA (sRNA), respectively, are shown, wherein the protein expression levels were analyzed by SDS-PAGE, quantified using a densitometer, and then averaged.

[0018] Figure 5 The results of SDS-PAGE analysis of the expression level of silk protein composed of 128 repeat sequences are shown. (Lane 1: marker showing the standard molecular weight of the protein; Lane 2: at an OD of 0.4 600 The results of protein expression induction in the strain transformed with plasmid pSH128; Lane 3: at OD 0.4 600 Results of protein expression induction in strains transformed with plasmids pSH128 and pACYC184-rnpA(sRNA).

[0019] Figure 6 This is a diagram showing the results of examining whether the intracellular mRNA level is increased by reducing the expression of the rnpA gene.

[0020] Figure 7is a graph showing the amount of silk protein composed of 96 repeat sequences prepared by fed-batch culture ( Figure 7 a), and display the electrophoresis results of the protein ( Figure 7 b).

[0021] Figure 8 The results of electrophoresis performed using the system for reducing rnpA gene expression of the present invention are shown to confirm that the expression of difficult-to-express proteins other than silk proteins is increased. Specifically, Figure 8 a shows the results of SDS-PAGE analysis of malic enzyme (SfcA) expression levels. (Lane 1: marker showing the standard molecular weight of the protein; Lane 2: the result of protein expression induced in non-transformed BL21 (DE3); Lanes 3 and 4: at an OD of 0.4 600 The results of protein expression induction in the strain transformed with SfcA; Lanes 5 and 6: at OD 0.4 600 Results of inducing protein expression in strains transformed with SfcA and rnpA (sRNA). Figure 8 b shows the results of Cat2 expression level analysis by SDS-PAGE. (Lane 1: marker showing the standard molecular weight of protein; Lanes 2 and 3: at OD 0.4 600 Results of protein expression induction in strains transformed with Cat2; Lanes 4 and 5: at OD 0.4 600 Results of inducing protein expression in strains transformed with Cat2-rnpA(sRNA). Figure 8 c shows the results of SDS-PAGE analysis of SrtA expression levels. (Lane 1: markers showing the standard molecular weight of proteins; Lanes 2 and 3: markers showing the expression of SrtA at an OD of 0.4 600 The results of protein expression induction in the strain transformed with SrtA; Lanes 4 and 5: at OD 0.4 600 Results of inducing protein expression in strains transformed with srtA-rnpA (sRNA). Figure 8 d shows the results of SDS-PAGE analysis of CYP73A5 expression levels. (Lane 1: markers showing the standard molecular weight of the protein; Lanes 2 and 3: markers showing the standard molecular weight of the protein at OD 0.4 600 Results of protein expression induction in strains transformed with CYP73A5; Lanes 4 and 5: at OD 0.4 600 Results of inducing protein expression in strains transformed with CYP73A5-rnpA(sRNA). Figure 8 e shows the results of SDS-PAGE analysis of CYP98A3 expression levels. (Lane 1: markers showing the standard molecular weight of the protein; Lanes 2 and 3: markers showing the expression of CYP98A3 at an OD of 0.4 600Results of protein expression induction in strains transformed with CYP98A3; Lanes 4 and 5: at OD 0.4 600 Results of inducing protein expression in strains transformed with CYP98A3-rnpA(sRNA).

[0022] Best Mode for Carrying Out the Invention

[0023] The present invention has developed a method for improving the expression of difficult-to-express recombinant proteins that are difficult to prepare in the prior art by reducing the expression of ribonuclease P in a recombinant microorganism to increase the mRNA level of the useful protein in the cell.

[0024] In the present invention, the expression of the rnpA gene, which is a component of ribonuclease P, was reduced using an sRNA system comprising sRNA, and as a result, the expression of high molecular weight silk protein, which is a difficult-to-express protein, was shown to be significantly increased.

[0025] The term "difficult-to-express protein" as used in the present invention refers to a protein having a molecular weight of 50 kDa or greater.

[0026] Therefore, on the one hand, the present invention relates to a recombinant vector for expressing a target protein, the recombinant vector comprising a gene encoding the target protein and an sRNA against a gene encoding ribonuclease P, and to a recombinant microorganism transformed with the recombinant vector.

[0027] In the present invention, the target protein may be a protein selected from the group consisting of difficult-to-express proteins, silk proteins, antibodies, enzymes, cytochromes, and transpeptidase A, but is not limited thereto.

[0028] In the present invention, the sRNA may be an sRNA targeting the rnpA gene, and may have a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3, but is not limited thereto, as long as it reduces the expression of the rnpA gene.

[0029] In the present invention, examples of microorganisms for preparing proteins that can be used include Escherichia, Pseudomonas, Saccharomyces, etc., preferably Escherichia microorganisms, most preferably Escherichia coli. Specifically, it is advantageous that Escherichia coli can be easily industrialized because its gene information and culture conditions widely used in industry are well known.

[0030] In one embodiment of the invention, a recombinant E. coli strain is constructed by using a gene encoding a silk protein produced by a dragline silk protein obtained from a golden silk spider (Nephila clavipes), a nucleotide sequence encoding a glycine tRNA, and a sRNA conversion that reduces the expression of RnpA as a ribonuclease P component. The constructed recombinant E. coli strain is cultured. As a result, it can be seen that the protein of the silk protein increases by 3 times or more. In addition, it is shown that the expression of a silk protein composed of a repetitive sequence of 16, 32, 48, 64, 80, 96, 112 and 128 specific amino acid sequences (SGRGGLGGTGAGMAAAAAMGGAGQGGYGGLGSQG) by reducing the rnpA gene as a ribonuclease P component is significantly increased. Further, it is shown that by reducing the expression of the rnpA gene as a ribonuclease P component, the expression of eGFP, SfcA and full-length IgG antibodies is also significantly increased.

[0031] In view of the results showing that the production of long recombinant proteins or difficult-to-express proteins can be increased by reducing the expression of the rnpA gene as a component of ribonuclease P, it is obvious to those skilled in the art that degradation of messenger RNA (mRNA) has a great effect on protein expression, and overexpression of target proteins can be effectively achieved by reducing the expression of genes related to messenger RNA (mRNA) degradation.

[0032] In another embodiment of the present invention, the expression of a gene encoding malic enzyme (sfcA), a gene encoding transpeptidase A (srtA), a gene encoding 4-hydroxybutyrate coenzyme A transferase (Cat2), and a cytochrome P450 gene (CYP73A5; cinnamate 4-hydroxylase and CYP98A3) was detected using a system for reducing the expression of the rnpA gene of the present invention. As a result, it was shown that the strain expressing sRNA against the rnpA gene together with the gene encoding each protein showed a higher protein expression level than the strain not expressing sRNA ( Figure 8 ).

[0033] In another aspect, the present invention relates to a recombinant microorganism, into which a gene encoding a target protein and an sRNA targeting a gene encoding ribonuclease P are introduced.

[0034] In the present invention, the gene encoding the target protein and the sRNA against the gene encoding ribonuclease P may be present in separate vectors or may be incorporated into the chromosome of the microorganism.

[0035] In another aspect, the present invention relates to a method for preparing a difficult-to-express target protein, the method comprising the following steps: (a) preparing the target protein by culturing a recombinant microorganism and inducing expression of the target protein in the recombinant microorganism; and (b) recovering the prepared target protein.

[0036] In one embodiment of the present invention, a recombinant Escherichia coli strain is constructed by transformation with a gene encoding a silk protein, a nucleotide sequence encoding a glycine tRNA, and an sRNA that reduces the expression of the rnpA gene as a component of ribonuclease P. The constructed recombinant Escherichia coli is cultured. As a result, it can be seen that the protein of the silk protein increases by 3 times or more.

[0037] In another example of the present invention, it was shown that when a gene encoding eGFP protein, a gene encoding sfcA protein, and a gene encoding a full-length IgG antibody protein and sRNA against rnpA gene were co-expressed, the expression of these proteins increased.

[0038] In the present invention, preferably, as a difficult-to-express protein, the target protein may be a large protein having a molecular weight of 50 kDa or more. For example, the target protein may be a protein selected from the group consisting of silk protein, antibodies, enzymes, cytochromes, and transpeptidase A, but is not limited thereto.

[0039] In still another aspect, the present invention relates to a method for preparing a target protein, comprising the steps of: expressing and preparing the target protein by culturing a recombinant microorganism into which a gene encoding the target protein is introduced; and recovering the prepared target protein, wherein the expression of ribonuclease P in the recombinant microorganism is reduced to increase the expression of the target protein.

[0040] In the present invention, the target protein may be a difficult-to-express protein or a protein having a molecular weight of 50 kDa or more, and a substance that inhibits the expression of ribonuclease P may be added.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures described below are those well known and commonly used in the art.

[0042] The definitions of the main terms used in the detailed description of the present invention are as follows.

[0043] The term "sRNA (small RNA)" used in the present invention refers to a short-length RNA generally having a length of 200 nucleotides or less, which is not translated into protein and effectively inhibits the translation of a specific mRNA by complementary binding.

[0044] The term "ribosome binding site" used in the present invention refers to a site where ribosome binds to mRNA for transcribing the mRNA.

[0045] The term "gene" as used herein is intended to have the broadest meaning, and a gene may encode a structural protein or a regulatory protein. In this context, a regulatory protein includes a transcription factor, a heat shock protein, or a protein that participates in DNA / RNA replication, transcription, and / or translation. In addition, a target gene whose expression is to be suppressed may exist as an extrachromosomal element.

[0046] The term "vector" as used in the present invention refers to a DNA construct comprising a DNA sequence operably linked to a suitable control sequence, which is capable of affecting the expression of the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once introduced into a suitable host, the vector may replicate and function independently of the host genome, or in some cases integrate into the genome itself. In this article, "plasmid" and "vector" are sometimes used interchangeably, as "plasmid" is the most commonly used form of vector. For the purposes of the present invention, plasmid vectors are preferably used. Typical plasmid vectors that can be used for this purpose include: (a) an origin of replication, where replication occurs efficiently so that hundreds of plasmid vectors are produced per host cell; (b) an antibiotic resistance gene, by which host cells transformed with the plasmid vector can be selected; and (c) a restriction enzyme site, where exogenous DNA fragments can be inserted. Even if there are no suitable restriction enzyme sites in the vector, conventional synthetic oligonucleotide adapters or linkers are used to facilitate connection between the vector and the exogenous DNA fragment. After connection, the vector should be transformed into a suitable host cell. Transformation can be easily achieved by the calcium chloride method or electroporation (Neumann et al., EMBO J., 1:841, 1982). Expression vectors known in the art can be used as vectors for expressing the sRNA of the present invention.

[0047] When a nucleic acid sequence is configured to have a functional relationship with other nucleic acid sequences, this nucleic acid sequence is operably connected. The nucleotide sequence can be a gene and a connected control sequence, and the gene can be expressed when a suitable molecule (e.g., a transcriptional activator) binds to the control sequence. For example, when the DNA of a presequence or secretory leader sequence is expressed as a preprotein involved in the secretion of a polypeptide, it is operably connected to the DNA encoding the polypeptide; when a promoter or enhancer affects the transcription of a sequence, it is operably connected to the coding sequence; or when a ribosome binding site affects the transcription of a sequence, it is operably connected to the coding sequence, or when it is configured to facilitate translation, it is operably connected to the coding sequence. Generally, the term "operably connected" refers to that the DNA connection sequence is adjacent, and in the case of a secretory leader sequence, the DNA connection sequence is adjacent and present in the reading frame. However, enhancers are not necessarily adjacent. The connection between these sequences is carried out by connecting at a convenient restriction enzyme site. However, when this site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional methods.

[0048] In addition, the present invention relates to a recombinant microorganism into which an expression vector comprising a nucleic acid encoding sRNA is introduced. The term "transformation" as used in the present invention means that DNA can be replicated as an extrachromosomal factor or as a host by introducing DNA to complete the entire chromosome.

[0049] Of course, it should be understood that not all vectors and expression control sequences all play the same role of expressing the DNA sequence of the present invention. Similarly, not all hosts all play the same role for the same expression system. However, without departing from the scope of the present invention or not taking the additional experimental burden, those skilled in the art can appropriately make a selection from a variety of vectors, expression control sequences and hosts. For example, it is necessary to consider that the host selects the vector, because the vector must be replicated in the host. Specifically, the copy number of the vector, the ability to regulate the copy number and the expression of other proteins encoded by the corresponding vector (for example, the expression of antibiotic markers) should also be considered. Example

[0050] Hereinafter, the present invention is further described in detail with reference to examples. It will be apparent to those skilled in the art that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

[0051] Example 1: Construction of recombinant plasmid pTetgly2glyAN-rnpA (sRNA)

[0052] All genetic manipulation procedures followed standard methods (Sambrook et al., Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0053] In order to introduce the sRNA system into the pTetlgy2glyAN vector (Korean Patent No. 1147860), gene manipulation was performed, and in order to obtain the ribosome binding site involved in the translation of the rnpA gene, inverse PCR was performed using primers of SEQ ID NOs: 4 and 5, thereby obtaining rnpA sRNA (SEQ ID NO: 1).

[0054] SEQ ID NO:4:5'-cctgggaaatgcgagcttaaccactttctgttgggccattgcattg-3'

[0055] SEQ ID NO:5:5'-GCAACCATTATCACCGCCA-3

[0056] PCR reactions were performed using Pfu polymerase (SolGent, Korea) under the following conditions: initial denaturation at 95°C for 5 min, followed by 28 cycles, each cycle consisting of denaturation at 95°C for 30 s, annealing at 57°C for 180 s, and extension at 72°C for 60 s, followed by a final extension at 72°C for 5 min.

[0057] The PCR product was electrophoresed on an agarose gel to obtain a purified 5000-bp PCR product. The purified PCR product was incubated with restriction enzyme DpnI (New England Biolabs, USA) for 1 hour, and then ligated with pTetlgy2glyAN by T4 DNA ligase (Roche, Germany), and the resulting vector was transformed into Escherichia coli dH5α (FhuA2lac(del)U169phoAglnV44Φ80'lacZ(del)M15gyrA96recA1relA1endA1thi-1hsdR17, Invitrogen).

[0058] The transformed strain was screened on LB agar solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L and agar 15 g / L) containing 34 mg / L chloramphenicol, thereby obtaining the recombinant plasmid pTetgly2glyAN-rnpA (sRNA) ( Figure 1 The constructed recombinant plasmid was confirmed by restriction enzyme cleavage and DNA sequencing.

[0059] Example 2: Construction of a recombinant plasmid containing a gene encoding a high molecular weight silk protein

[0060] In order to construct a recombinant plasmid containing 32 gene repeat sequences encoding silk proteins produced by modifying the dragline silk protein (a difficult-to-express protein with a large size) derived from the golden silk spider, the plasmid pSH16a (Lee et al., Theories and Applications of Chem. Eng., 8:3969, 2002) consisting of 16 gene repeat sequences encoding silk proteins was digested with restriction enzymes SpeI and NheI to obtain a 1.7 kb fragment. This fragment was ligated to the plasmid pSH16a digested with restriction enzyme SpeI, thereby obtaining a recombinant plasmid pSH32. The orientation of the ligated insert sequence was determined by digestion with restriction enzymes SpeI and NheI (New England Biolabs, USA).

[0061] Similarly, plasmid pSH16a was digested with restriction enzymes SpeI and NheI to obtain a 1.7 kb fragment, which was then ligated with plasmid pSH32 digested with restriction enzyme SpeI to construct recombinant plasmid pSH48. Plasmid pSH32 was digested with restriction enzymes SpeI and NheI to obtain a 3.4 kb fragment, which was then ligated with plasmid pSH64 digested with restriction enzyme SpeI to construct recombinant plasmid pSH64. Plasmid pSH48 was digested with restriction enzymes SpeI and NheI to obtain a 5.1 kb fragment, which was then ligated with plasmid pSH32 digested with restriction enzyme SpeI to construct recombinant plasmid pSH80. Plasmid pSH16 was digested with restriction enzymes SpeI and NheI to obtain a 1.74 kb fragment, which was then ligated with plasmid pSH80 digested with restriction enzyme SpeI to construct recombinant plasmid pSH96. Plasmid pSH32 was digested with restriction enzymes SpeI and NheI to obtain a 3.4 kb fragment, which was then ligated with plasmid pSH80 digested with restriction enzyme SpeI to construct recombinant plasmid pSH112. Plasmid pSH64 was digested with restriction enzymes SpeI and NheI to obtain a 7.8 kb fragment, which was then ligated with plasmid pSH64 digested with restriction enzyme SpeI to construct recombinant plasmid pSH128. The orientation of each ligated insert was determined by using restriction enzymes SpeI and NheI.

[0062] Example 3: Examination of the increase in silk protein expression caused by reducing rnpA gene expression by sRNA system

[0063] To examine the effects of co-overexpression of the glycine tRNA gene and reduction of the expression of the ribonuclease P (rnpA) gene by using the sRNA system in the preparation of silk protein, the plasmid pTetgly2glyAN-rnpA(sRNA) obtained in Example 1 and plasmids pSH16, pSH32, pSH48, pSH64, pSH80, pSH96 and pSH112 containing 16, 32, 48, 64, 80, 96 and 112 gene repeat sequences encoding silk protein were used to transform Escherichia coli BL21(DE3) (F-ompT hsdSB(rB-mB-)gal dcm(DE3), a prophage carrying the T7 RNA polymerase gene) (New England Biolabs, USA), respectively.

[0064] E. coli BL21 (DE3) transformed with plasmids pACYC184 and pSH16, pSH32, pSH64, pSH96 and pSH128 was used as a control. Each transformed strain was inoculated into 10 ml of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L) (containing 34 mg / L chloramphenicol, 25 mg / L kanamycin and 1% arabinose) and cultured at 25 ° C and 220 rpm. Next, each strain was cultured at 37 ° C and 220 rpm under the above-mentioned culture medium conditions. When the culture reached an OD of 0.4, the culture was stirred for 24 hours. 600 1 mM IPTG was added to the culture medium to induce silk protein gene expression. After 4 hours of induction, the culture was sampled, centrifuged at 4°C and 10,000g for 10 minutes, and the obtained cell pellet was dissolved in TE buffer and 5× Laemmli sample buffer. The same amount of samples (0.024 mg) were separated using 10% SDS-PAGE gel, and then stained with Coomassie Brilliant Blue R250 (Bio-Rad, USA) solution and quantified using a GS-710 calibrated imaging densitometer (Bio-Rad, USA) ( Figure 2 to 4).

[0065] In addition, at an OD of 0.4 600 Expression of strains transformed with plasmids pSH96 and pTetgly2glyAN-rnpA (sRNA) was induced, and protein expression was analyzed by SDS-PAGE, quantified using a densitometer, and averaged. The results are shown in Figure 4b In addition, the results of expression using plasmid pSH128 are shown in Figure 5 .

[0066] As a result, it can be seen that due to the reduced expression of ribonuclease P (RnpA), the expression of silk protein composed of 16 repeat sequences increased by about 150% relative to the control, and the expression of silk protein composed of 32 repeat sequences increased by about 300% relative to the control. In addition, the expression of silk protein composed of 48 repeat sequences increased by about 300%, and the expression of silk protein composed of 64 repeat sequences increased by about 300%. In addition, the expression of silk protein composed of 80 repeat sequences increased by about 300%, the expression of silk protein composed of 112 repeat sequences increased by about 200%, and the expression of silk protein composed of 128 repeat sequences increased by about 150%.

[0067] Example 4: Examination of the increase in mRNA levels caused by reducing rnpA gene expression by the sRNA system

[0068] In this example, in order to prevent messenger RNA degradation in the preparation of recombinant proteins, difficult-to-express proteins, and useful proteins, the expression of the rnpA gene, which is a component of ribonuclease P, was reduced by introducing an sRNA system. In addition, it was examined whether the intracellular mRNA level would increase by reducing the expression of the rnpA gene.

[0069] Specifically, the plasmid pTetgly2glyAN-rnpA(sRNA) and pSH32 obtained in Example 1 were used to transform Escherichia coli BL21(DE3) (F-ompT hsdSB(rB-mB-)gal dcm(DE3), a bacteriophage carrying the T7 RNA polymerase gene) (New England Biolabs, USA).

[0070] E. coli BL21 (DE3) transformed with plasmids pACYC184 and pSH32 was used as a control. Each transformed strain was inoculated into 10 ml of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L) (containing 34 mg / L chloramphenicol, 25 mg / L kanamycin and 1% arabinose) and cultured at 25°C and 220 rpm. Next, each strain was cultured at 37°C and 220 rpm under the above culture medium conditions. When the culture reached an OD of 0.4, the culture was stirred for 24 h. 600 1 mM IPTG was added to the culture medium to induce silk protein gene expression. After 4 hours of induction, the culture was sampled, centrifuged at 4°C and 10,000 g for 10 minutes to obtain cell pellets, and RNA was extracted from the cell pellets.

[0071] For RNA extraction, 1ml Trizol was added to the cell pellet, and then the cell pellet solution was stirred for 1 minute and allowed to stand at room temperature for 5 minutes. Next, chloroform in an amount equal to 20% of the total amount was added to the cell pellet solution, then stirred for 15 seconds and centrifuged at 13,000rpm for 15 minutes. The prepared supernatant was transferred to a new test tube, and the same amount of isopropanol was added thereto and carefully stirred. Then, the mixture was centrifuged at 13,000rpm for 30 minutes and the supernatant was removed. Next, 1ml 70% ethanol was added to the remaining material, and then centrifuged at 13,000rpm for 5 minutes, and the above procedure was repeated again. Next, the resulting material was dried at room temperature and dissolved in 30μl of RNase-free distilled water. In order to use complementary DNA (cDNA) instead of the extracted RNA, qPCR was performed using Rocketstrip (Bioneer, Korea). The extracted RNA was adjusted to 500ng, and 2μ1 DN9 primers were added thereto. Then, the reaction mixture was adjusted to a total volume of 20μl using RNase-free distilled water. The PCR reaction was performed under the following conditions: initial annealing at 30°C for 150 seconds, cDNA synthesis at 60°C for 1 hour, and then heat inactivation at 95°C for 300 seconds.

[0072] In order to perform RT-PCR using the prepared cDNA, a reaction mixture containing 8 μl of RNase-free distilled water, 10 μl of SYBR Green Mastermix (Enzynomics, Korea), 1 μl of 1 / 10 diluted cDNA and 0.5 μl of each primer in a total volume of 20 μl was used. The RT-PCR reaction was performed under the following conditions: initial activation at 95°C for 10 minutes, followed by 45 cycles, each cycle consisting of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30 seconds. In order to determine the melting curve, the temperature was increased by 0.5°C from 55°C to 95°C, and the reaction mixture was kept at each temperature for 5 seconds.

[0073] The result is that, Figure 6 As shown, in the case of cells in which ribonuclease P (RnpA) expression was reduced, the level of mRNA was 24 times higher than that of the control. This suggests that degradation of mRNA is prevented by reducing ribonuclease P (RnpA) expression.

[0074] Example 5: Examination of increased spider silk protein production by fed-batch fermentation

[0075] The strain transformed with plasmids pSH96 and pACYC184-rnpA (sRNA) constructed in Example 3 was cultured by fed-batch fermentation to examine the increase in protein production in the strain.

[0076] For fed-batch fermentation, a 6.6 L fermentor (Bioflo 3000; New Brunswick Scientific Co.) was used, and 1.6LR / 2 medium, 10 g / L glucose, 0.7 g / L MgSO4·7H2O, and antibiotics (50 μg / mL kanamycin and / or 35 μg / mL chloramphenicol) were added to the strain in the fermentor. To adjust the dissolved oxygen level to 40%, the air saturation was adjusted while increasing the stirring speed to 1000 rpm. The feed solution consisted of 700 g / L glucose and 20 g / L MgSO4·7H2O, and the pH of the culture was adjusted using 28% (v / v) ammonia solution. When OD 600 When the protein expression was induced, 1 mM IPTG was added to the culture. After 8 hours of induction, the culture was sampled at 2 hour intervals. The cell proliferation and protein concentration obtained are shown in Figure 7 .like Figure 7 As shown, a protein concentration of 0.9 g / L can be obtained, which is 30% higher than the previously known concentration (0.7 g / L).

[0077] Example 6: Examination of increasing the expression of other proteins by reducing rnpA gene expression

[0078] Using the system for reducing rnpA gene expression of the present invention, the expression of genes encoding malic enzyme (sfcA, SEQ ID NO: 6), transpeptidase A (srtA, SEQ ID NO: 7), 4-hydroxybutyrate coenzyme A transferase (Cat2, SEQ ID NO: 8), and cytochrome P450 genes (CYP73A5; cinnamate 4-hydroxylase: SEQ ID NO: 9 and CYP98A3: SEQ ID NO: 10) were analyzed. The transpeptidase A gene was synthesized by Bioneer (Korea), and the sfcA, cat2, CYP73A5, and CYP98A3 genes were obtained by PCR using the following primers.

[0079] sfcA_F:5'-CCATGGATATTCAAAAAAGAGTGAGT-3'(SEQ ID NO:11)

[0080] sfcA_R:5'-TCTAGATTAGATGGAGGTACGGCGGTA-3'(SEQ ID NO:12)

[0081] Cat2_F:5'-GAATTCATGGAGTGGGAAGAGATATATA–3'(SEQ ID NO:13)

[0082] Cat2_R:5'-GGTACCCTAAAATCTCTTTTTAAATTCATT-3'(SEQ ID NO:14)

[0083] CYP73A5_F:5'-GCGAAGCTTACAGTTCCTTGGTTTCATAA-3'(SEQ ID NO:15)

[0084] CYP73A5_R:5'-GTACATATGATGGACCTCCTCTTGCTGG-3'(SEQ ID NO:16)

[0085] CYP98A3_F:5'-GGATCCATGTCGTGGTTTCTAATAGC–3'(SEQ ID NO:17)

[0086] CYP98A3_R:5'-GAATTCTTACATATCGTAAGGCACGC-3'(SEQ ID NO:18)

[0087] RnpA sRNA (SEQ ID NO: 1) and each gene in sfcA, srtA and cat2 genes were inserted into pET30a (+) (Addgene, USA) and pTac15k (Addgene, USA) to obtain recombinant vectors. Each vector was transformed into Escherichia coli BL21 (DE3) strain. The strain transformed by inserting each gene in sfcA, srtA and cat2 genes into pET30a and pTac15k was used as a control.

[0088] Each transformed strain was inoculated into 10 ml LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L and NaCl 5 g / L) (containing 34 mg / L chloramphenicol, 25 mg / L kanamycin and 1% arabinose) and cultured at 25°C and 220 rpm. Next, each strain was cultured at 37°C and 220 rpm under the above culture conditions. When the culture reached an OD of 0.4, the culture was stirred for 2 h. 600 When 1 mM IPTG was added to the culture medium to induce the expression of each protein. After 4 hours of induction, the culture was sampled and centrifuged at 4°C and 10,000g for 10 minutes to obtain a cell pellet. The cell pellet was dissolved in TE buffer and 5×Laemmli sample buffer. The same amount of sample (0.024 mg) was separated on a 10% SDS-PAGE gel and then stained with Coomassie Brilliant Blue R250 (Bio-Rad, USA) solution.

[0089] The result is that, Figure 8As shown in , the expression levels of malic enzyme, transpeptidase A, 4-hydroxybutyrate CoA transferase (Cat2), cytochrome P450 (cinnamate 4-hydroxylase and CYP98A3) expressed together with RnpA sRNA were significantly higher than the protein expression levels in the absence of RnpA sRNA.

[0090] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that these descriptions are only for preferred embodiments and do not limit the scope of the present invention. Therefore, the actual scope of the present invention is defined by the appended claims and their equivalents.

[0091] Industrial Applicability

[0092] According to the present invention, the expression of a target protein, especially a difficult-to-express protein with a high molecular weight, can be significantly increased by reducing the expression of the rnpA gene, so that the present invention can be used to increase the yield of the protein.

Claims

1. A recombinant vector for expressing a target protein in Escherichia coli, the recombinant vector comprising a gene encoding the target protein and an sRNA targeting a gene encoding ribonuclease P, wherein the nucleotide sequence of the sRNA is SEQ ID NO:

1.

2. The recombinant vector of claim 1, wherein the target protein is a protein having a molecular weight of 50 kDa or greater. The recombinant vector of claim 1 , wherein the sRNA is directed against the rnpA gene.

4. The recombinant vector of claim 2, wherein the target protein is selected from the group consisting of silk protein, antibody, cytochrome, enzyme and transpeptidase A.

5. A recombinant Escherichia coli, into which the recombinant vector according to claim 1 is introduced.

6. A recombinant Escherichia coli, into which a gene encoding a target protein and an sRNA targeting a gene encoding ribonuclease P are introduced, wherein the nucleotide sequence of the sRNA is SEQ ID NO:

1.

7. The recombinant Escherichia coli of claim 6, wherein the target protein is a protein having a molecular weight of 50 kDa or more. The recombinant Escherichia coli according to claim 6 , wherein the sRNA is an sRNA against the rnpA gene.

9. A method for preparing a target protein, the method comprising the following steps: (a) expressing the target protein in the recombinant Escherichia coli by culturing the recombinant Escherichia coli of claim 5 or 6; and (b) Recovering the expressed target protein.

10. The method of claim 9, wherein the target protein is a protein having a molecular weight of 50 kDa or greater.

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