Method for preparing klentaq polymerase in pichia pastoris

Abstract

The present disclosure relates to a novel method for preparing KlenTaq polymerase lacking 5' to 3' exonuclease activity, and more specifically, to a method for preparing KlenTaq polymerase in Pichia pastoris, KlenTaq polymerase produced thereby, and a method for amplifying a target nucleic acid in a sample using the same. The KlenTaq polymerase produced from transformed Pichia pastoris according to the method of the present invention can prevent contamination of genomic DNA of E. coli, thereby reducing false positives in PCR for detecting urinary tract infection (UTI) causative agents including E. coli.

Description

METHOD FOR PREPARING KLENTAQ POLYMERASE IN PICHIA PASTORIS

[0001] Cross-Reference To Related Applications

[0002] This application claims priority from Korean Patent Application No. 10-2024-0068071, filed on May 24, 2024, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Technical Field

[0004] The present disclosure relates to a novel method for preparing KlenTaq polymerase lacking 5' to 3' exonuclease activity, and more specifically, to a method for preparing KlenTaq polymerase inPichia pastoris, KlenTaq polymerase produced thereby, and a method for amplifying a target nucleic acid in a sample using the same.

[0005]

[0006] Nucleic acid amplification is mainly used in molecular biology and biotechnology to detect and analyze a small quantity of target nucleic acids in a sample. For nucleic acid amplification, PCR (polymerase chain reaction) has been widely used, which involves repeating a series of steps of denaturing double-stranded DNAs into single-stranded DNAs, hybridizing primers with the single-stranded DNAs, and then extending the primers along the single-stranded DNAs by DNA polymerase.

[0007] DNA polymerase (deoxyribonucleic acid polymerase, E.C. number 2.7.7.7) as a key component in PCR plays a role in synthesizing DNA in the 5' to 3' direction depending on template DNA. Since PCR is performed at a high temperature of 50℃ or higher, the use of thermostable DNA polymerase is required. As a representative thermostable DNA polymerase,TaqDNA polymerase having a molecular weight of about 94 kDa, isolated from a thermophilic bacterium,Thermus aquaticusYT-1, has been widely used (Ishino, Y., et al., 1994).

[0008] On the other hand, KlenTaq polymerase (also called large fragment ofTaqDNA polymerase) is widely used instead of or in addition toTaqDNA polymerase.

[0009] KlenTaq polymerase is a truncated version ofTaqDNA polymerase that lacks its first 280 amino acids, named after the Klenow fragment. KlenTaq polymerase catalyzes the polymerization of nucleotides into duplex DNA in the 5' to 3' direction in the presence of magnesium, but lacks 5' to 3' exonuclease activity ofTaqDNA polymerase. Previous studies have shown that KlenTaq polymerase exhibits improved accuracy and thermostability compared toTaqDNA polymerase, and its PCR error rate is also about twice as low as that ofTaqDNA polymerase (Lawyer et al., J. Biol. Chem., 264(11), 6427-6437 (1989); Barnes, W. M. (1992). Gene, 112(1), 29-35). In addition, as KlenTaq polymerase is known to tolerate a wide range of magnesium concentrations, optimization of magnesium concentration is not necessary.

[0010] KlenTaq polymerase is typically produced by culture and purification using a recombinantE. coliexpression system. However, due to its powerful DNA-binding properties, KlenTaq polymerase is likely to bind to genomic DNA of recombinantE. coliduring its purification. Therefore, commercially available KlenTaq polymerases are known to be contaminated withE. coliDNA.

[0011] The use of KlenTaq polymerases contaminated withE. coliDNA in an amplification reaction (e.g., PCR) for detecting a target nucleic acid fromE. colimay lead to amplification of the contaminatedE. coliDNA and thus false positives.

[0012] In particular, urinary tract infection (UTI) is common in adults and even children, and more than 85% of cases are caused by bacteria includingE. coli. Recently, a method has been used as a standard for urinary tract infection tests to easily identify which strain has been infected by amplifying target DNA of various strains includingE. colithat easily infected with the urethra by PCR.

[0013] Therefore, there remains a need in the art to prepare KlenTaq polymerase not contaminated withE. coliDNA.

[0014]

[0015] In addition toE. coli, yeasts such asSaccharomyces cerevisiaehave been widely used to express heterologous proteins. Recently,Pichia pastorishas been attracting attention.

[0016] P. pastoriscan be engineered as easily asE. coliorS. cerevisiae, and unlikeE. coli, it has an expression system unique to eukaryotic cells, including processing, folding, and post-translational modification of proteins. BothP. pastorisandS. cerevisiaehave some eukaryotic and prokaryotic characteristics, but the proteins expressed inP. pastorishave conformations that are closer to humans and animals than those expressed inS. cerevisiae. Thus,P. pastorishas an advantage overS. cerevisiaein producing proteins that will actually act on animal cells. Also,P. pastorisis advantageous in that it produces a higher amount of soluble protein per the same cell mass thanS. cerevisiae.

[0017] However, no attempt has been made to prepare KlenTaq polymerase inPichia pastoris.

[0018] Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entirety are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

[0019]

[0020] The present inventors have endeavored to develop a method for preparing KlenTaq polymerase inPichia pastoris. As a result, the present inventors have developed a method for preparing KlenTaq polymerase not contaminated with nucleic acids derived fromE. coli, comprising incorporating a polynucleotide encoding the KlenTaq polymerase into an expression vector lacking a secretion signal peptide, transformingP. pastoriswith the expression vector, culturing the transformedP. pastoris, lysing the culturedP. pastoris, and purifying the KlenTaq polymerase from the lysate.

[0021] Thus, it is an object of the present disclosure to provide a method for preparing KlenTaq polymerase lacking 5' to 3' exonuclease activity.

[0022] It is another object of the present disclosure to provide KlenTaq polymerase prepared by the method as described above.

[0023] It is still another object of the present disclosure to provide a method for amplifying a target nucleic acid in a sample using the KlenTaq polymerase as described above.

[0024] It is still another object of this disclosure to provide a transformedPichia pastorisfor preparing the KlenTaq polymerase as described above.

[0025]

[0026] In an aspect of the present disclosure, there is provided a method for preparing KlenTaq polymerase lacking 5' to 3' exonuclease activity, comprising: (a) transformingPichia pastoriswith an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the KlenTaq polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide; (b) culturing the transformedP. pastorisin a culture medium under conditions permitting intracellular expression of the KlenTaq polymerase; and (c) lysing the culturedP. pastorisand purifying the intracellularly expressed KlenTaq polymerase from the lysate.

[0027] In certain embodiments, the polynucleotide encoding the KlenTaq polymerase is codon-optimized forP. pastoris.

[0028] In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises a sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto.

[0029] In certain embodiments, the promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

[0030] In certain embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a histidine tag, which is linked to the terminus of the polynucleotide encoding the KlenTaq polymerase.

[0031] In certain embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a (GSG)nlinker between the polynucleotide encoding the KlenTaq polymerase and the nucleic acid sequence encoding the histidine tag, wherein n is an integer from 1 to 5, G denotes glycine, and S denotes serine.

[0032] In certain embodiments, the expression vector further comprises an antibiotic resistance gene as a selectable marker.

[0033] In certain embodiments, the antibiotic resistance gene is a bleomycin resistance gene, a phleomycin resistance gene, or a zeocin resistance gene.

[0034] In certain embodiments, the expression vector comprises a plurality of the nucleic acid constructs.

[0035] In certain embodiments, the plurality of the nucleic acid constructs are composed of 6 to 16 nucleic acid constructs.

[0036] In certain embodiments, the method further comprises linearizing the expression vector prior to the transformation of step (a).

[0037] In certain embodiments, the transformation of step (a) induces homologous recombination between the expression vector and the genome ofP. pastoris.

[0038] In certain embodiments, the purified, intracellularly expressed KlenTaq polymerase is produced in an amount of 0.1 to 1.5 mg per gram of cells ofP. pastoris.

[0039] In certain embodiments, the purified, intracellularly expressed KlenTaq polymerase is free of nucleic acids derived fromE. coli.

[0040] In certain embodiments, the purified, intracellularly expressed KlenTaq polymerase does not lead to any false positives from a non-targetE. coliin an amplification reaction.

[0041] In another aspect of the present disclosure, there is provided KlenTaq polymerase, prepared by the method as described above.

[0042] In certain embodiments, the KlenTaq polymerase is free of nucleic acids derived fromE. coli.

[0043] In certain embodiments, the KlenTaq polymerase does not lead to any false positives from a non-targetE. coliin an amplification reaction

[0044] In another aspect of the present disclosure, there is provided a method of amplifying a target nucleic acid in a sample using the KlenTaq polymerase as describe above.

[0045] In certain embodiments, the target nucleic acid is fromE. coli.

[0046] In another aspect of the present disclosure, there is provided a transformedPichia pastoris, prepared by transformingP. pastoriswith an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding a KlenTaq polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.

[0047]

[0048] The features and advantages of the present disclosure are summarized as follows:

[0049] (a) Conventional methods for producing a heterologous protein in a transformedPichia pastorisrequire use of highly flammable and toxic methanol to induce promoter transcription, whereas the method of the present disclosure can avoid the use of methanol, enabling safer production of the heterologous protein.

[0050] (b) KlenTaq polymerase produced byE. coliexpression systems may be contaminated byE. coligenomic DNA and thus lead to false positives due to non-target nucleic acids from the contaminatedE. coliin amplification of target nucleic acids fromE. coli, whereas KlenTaq polymerase prepared by the method of the present disclosure is free of any nucleic acids fromE. coli, thereby preventing false positives.

[0051] (c) The use of an expression vector containing a plurality of nucleic acid constructs according to the method of the present disclosure can further increase the production yield of KlenTaq polymerase.

[0052]

[0053] Fig. 1 depicts a map of a commercially available expression vector, "pGAPZαA", which is used to prepare an expression vector according to an embodiment of the present disclosure.

[0054] Fig. 2 shows the electrophoresis results of pGAPZ KlenTaq pol 2M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second lane represents the result of pGAPZ KlenTaq pol for comparison; and the third to seventh lanes represent the results of pGAPZ KlenTaq pol 2M obtained from five transformants.

[0055] Fig. 3 shows the electrophoresis results of pGAPZ KlenTaq pol 4M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second lane represents the result of pGAPZ KlenTaq pol 2M for comparison; and the third to seventh lanes represent the results of pGAPZ KlenTaq pol 4M obtained from five transformants.

[0056] Fig. 4 shows the electrophoresis results of pGAPZ KlenTaq pol 8M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second lane represents the result of pGAPZ KlenTaq pol 4M for comparison; and the third to seventh lanes represent the results of pGAPZ KlenTaq pol 8M obtained from five transformants.

[0057] Fig. 5 shows the electrophoresis results of pGAPZ KlenTaq pol 12M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second and third lanes represent the results of pGAPZ KlenTaq pol 12M obtained from two transformants; and the fourth lane represents the result of pGAPZ KlenTaq pol 8M for comparison.

[0058] Fig. 6 shows the SDS-PAGE results of cell lysates ofPichia pastoristransformed with pGAPZ KlenTaq pol 8M according to the present disclosure. The first lane represents a marker; the second lane represents the result for cell lysates of untransformedPichia pastorisX-33 for comparison, and the third to tenth lanes represent the results for cell lysates ofPichia pastorisX-33 transformed with pGAPZ KlenTaq pol 8M.

[0059] Fig. 7 shows the SDS-PAGE results of cell lysates ofPichia pastoristransformed with pGAPZ KlenTaq pol 12M according to the present disclosure. The first lane represents a DNA ladder marker; the second to tenth lanes represent the results for cell lysates ofPichia pastorisX-33 transformed with pGAPZ KlenTaq pol 12M.

[0060] Fig. 8 shows the SDS-PAGE results of cell lysates ofPichia pastoristransformed with pGAPZ KlenTaq pol 8M, cultured for different times (23 h, 28.5 h, 31.5 h, and 48 h).

[0061] Fig. 9 shows the SDS-PAGE results of cell lysates ofPichia pastoristransformed with pGAPZ KlenTaq pol 12M, cultured for different times (23 h, 28.5 h, 31.5 h, and 48 h).

[0062] Fig. 10 shows the electrophoresis results of PCR products using KlenTaq polymerase, which was produced fromPichia pastoristransformed with pGAPZ KlenTaq pol 12M. The first lane represents a DNA ladder marker; the second lane represents the result of PCR products using 1 μg of KlenTaq polymerase obtained in Example 3; the third lane represents the result of PCR products using 0.5 μg of wild-typeTaqpolymerase; and the fourth lane represents the result of PCR products using 1 μg of wild-typeTaqpolymerase.

[0063] Fig. 11A shows fluorescence measured at 30 second intervals during a reaction of EvaEZTMreagent and 3.125 ng, 1.563 ng, 0.781 ng, or 0 ng of the KlenTaq polymerase according to the present disclosure at 72°C for 60 minutes, in order to determine the specific activity of the KlenTaq polymerase according to the present disclosure.

[0064] Fig. 11B shows the slope calculated from the data showing the initial linear function in the plot of Fig. 11A, in order to determine the specific activity of the KlenTaq polymerase according to the present disclosure. The x-axis represents the concentration of KlenTaq polymerase, and the y-axis represents the amount of dNTP consumed.

[0065] Fig. 12 shows the amplification of targetE. coligenomic DNA using KlenTaq polymerase according to the present disclosure. The first lane represents a DNA ladder marker; the second lane represents the result of PCR using a 1 / 2 dilution of KlenTaq polymerase according to the present disclosure in the presence ofE. coligenomic DNA; the third lane represents the result of PCR using a 1 / 2 dilution of KlenTaq polymerase according to the present disclosure in the absence ofE. coligenomic DNA; the fourth lane represents the result of PCR using a 1 / 5 dilution of KlenTaq polymerase according to the present disclosure in the presence ofE. coligenomic DNA; the fifth lane represents the result of PCR using a 1 / 5 dilution of KlenTaq polymerase according to the present disclosure in the absence ofE. coligenomic DNA; the sixth lane represents the result of PCR using a 1 / 10 dilution of KlenTaq polymerase according to the present disclosure in the presence ofE. coligenomic DNA; and the seventh lane represents the result of PCR using a 1 / 10 dilution of KlenTaq polymerase according to the present disclosure in the absence ofE. coligenomic DNA.

[0066]

[0067] I. Method for preparing KlenTaq polymerase

[0068] In an aspect of the present disclosure, there is provided a method for preparing KlenTaq polymerase lacking 5' to 3' exonuclease activity, comprising:

[0069] (a) transformingPichia pastoriswith an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the KlenTaq polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide;

[0070] (b) culturing the transformedP. pastorisin a culture medium under conditions permitting intracellular expression of the KlenTaq polymerase; and

[0071] (c) lysing the culturedP. pastorisand purifying the intracellularly expressed KlenTaq polymerase from the lysate.

[0072] Hereinafter, the steps of the method according to the present disclosure is described in detail.

[0073]

[0074] Step (a): Transforming Pichia Pastoris with Expression Vector

[0075] In step (a) of the method of the present disclosure,Pichia pastorisis transformed with an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the KlenTaq polymerase, and (ii) a promoter operably linked thereto.

[0076]

[0077] KlenTaq polymerase

[0078] As used herein, the term "KlenTaq polymerase" or "KlenTaq DNA polymerase" refers to a truncated version lacking the first 280 amino acids of DNA polymerase, which is derived from the thermophilic eubacteriumThermus aquaticusYT-1. KlenTaq polymerase, also called the large fragment ofTaqDNA polymerase or KlenowTaqDNA polymerase, lacks 5' to 3' exonuclease activity ofTaqDNA polymerase. KlenTaq polymerase is known to exhibit improved accuracy and thermostability compared toTaqDNA polymerase, and its PCR error rate is also about twice as low as that ofTaqDNA polymerase.

[0079] KlenTaq polymerase is widely used for nucleic acid amplification reactions, such as real-time PCR, instead of or in addition toTaqDNA polymerase.

[0080] The KlenTaq polymerase of the present disclosure may be wild-type or a variant thereof.

[0081] Wild-type KlenTaq polymerase or variants thereof are well known in the art.

[0082] Wild-type KlenTaq polymerase is an N-terminal deletion of 280 amino acids ofTaqDNA polymerase, which consists of 832 amino acids, as can be identified in GenBank Accession no. J04639.1. Wild-type KlenTaq polymerase is also called KlenTaq1 (Barnes, 1994; U.S. Patent No. 5,436,149).

[0083] The term "variant" as used herein refers to a KlenTaq polymerase having substitution, insertion, or deletion of amino acid(s) relative to the wild-type KlenTaq polymerase, for the purpose of improving the function of wild-type KlenTaq polymerase. The variants of KlenTaq polymerase may have a sequence identity of at least 90%,e.g., at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% to the nucleotide sequence of the wild-type KlenTaq polymerase. Examples of variants of KlenTaq polymerase are found in Blatter, N, et al., (2013), Angew. Chem. Int. Ed. 52, 11935-11939; M. Drum et al., (2014), Plos One. 2014 May 6; 9(5):e96640; Barnes, W. M et al., (2021), Front. Bioeng. Biotechnol. 8:553474.

[0084]

[0085] Polynucleotide encoding KlenTaq polymerase

[0086] In certain embodiments, the polynucleotide encoding the KlenTaq polymerase is codon-optimized forP. pastoris.

[0087] As used herein, the term "codon optimization" or "codon-optimized" refers to a process of changing the codons of a heterologous protein based on codon usage bias of a host organism being transformed to increase expression of the heterologous protein. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase is codon-optimized by changing their codons based on codon usage bias ofP. pastoris. The sequence of the codon-optimized polynucleotide is different from that of the polynucleotide before codon-optimization, but their amino acid sequences are the same.

[0088] The codon usage bias ofP. pastoriscan be found,e.g., in a codon usage table, known in the art. For example, a codon usage table forP. pastorisknown in the art is as follows:

[0089] Amino acidCodonUsageAmino acidCodonUsageFUUU0.54YUAU0.47UUC0.46UAC0.53LUUA0.16STOPUAA0.51UUG0.33UAG0.29CUU0.16HCAU0.57CUC0.08CAC0.43CUA0.11QCAA0.61CUG0.16CAG0.39IAUU0.50NAAU0.48AUC0.31AAC0.52AUA0.18KAAA0.47MAUA1.00AAG0.53VGUU0.42DGAU0.58GUC0.23GAC0.42GUA0.15EGAA0.56GUG0.19GAG0.44SUCU0.29CUGU0.64UCC0.20UGC0.36UCA0.18STOPUGA0.20UCG0.09WUGG1.00PCCU0.35RCGU0.17CCC0.15CGC0.05CCA0.42CGA0.10CCG0.09CGG0.05TACU0.40SAGU0.15ACC0.26AGC0.09ACA0.24RAGA0.48ACG0.11AGG0.16AGCU0.45GGGU0.44GCC0.26GGC0.14GCA0.23GGA0.33GCG0.06GGG0.10

[0090] In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises a sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises or consists of a sequence of SEQ ID NO: 1. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises or consists of a sequence having at least 95% sequence identity to SEQ ID NO: 1. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises or consists of a sequence having at least 96% sequence identity to SEQ ID NO: 1. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises or consists of a sequence having at least 97% sequence identity to SEQ ID NO: 1. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises or consists of a sequence having at least 98% sequence identity to SEQ ID NO: 1. In certain embodiments, the polynucleotide encoding the KlenTaq polymerase comprises or consists of a sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0091] The nucleotide sequence of SEQ ID NO: 1 is as follows:

[0092]

[0093] (SEQ ID NO: 1)

[0094]

[0095] Nucleic acid construct

[0096] As used herein, the term "nucleic acid construct" refers to an artificially designed segment of DNA containing all the elements required for self-expression. The nucleic acid construct can typically include a promoter, a transcriptional terminator, a ribosome binding site, and a translation terminator, operably linked to transgenic genes. The nucleic acid construct may be in the form of a self-replicable expression vector.

[0097] In an embodiment, the nucleic acid construct herein comprises (i) a polynucleotide encoding the KlenTaq polymerase, and (ii) a promoter operably linked thereto.

[0098] As used herein, the term "promoter" refers to a nucleic acid sequence encoding amino acids containing a binding site for RNA polymerase and having activity of initiating transcription of downstream genes into mRNA.

[0099] The promoter as used herein includes various promoters known to be capable of driving the transcription of proteins of interest inP. pastoris. The promoter may be an inducible promoter or a constitutive promoter, depending on the expression pattern of the protein of interest. Examples include, without limitation, AOX1 (alcohol oxidase 1) promoter (Lin-Cereghino J, et al (2000) FEMS Microbiol Rev24:45-66), ADH3 (alcohol dehydrogenase) promoter (Karaoglan M, et al (2016) Protein Expr Purif 121:112-117), DAS (Dihydroxyacetone hormone) promoter (Tschopp JF, et al (1987) Nucleic Acids Res 15:3859-3876), FLD1 (Formaldehyde dehydrogenase) (Shen S, et al (1998) Gene 216:93-102), PEX8 (Peroxisomal matrix protein) promoter (Lin-Cereghino J, et al (2000) FEMS Microbiol Rev 24:45-66), ICL1 (Isocitrate lyase) promoter (Menendez J, et al (2003) Yeast 20:1097-1108), LRA3 (L-rhamnonate dehydratase) promoter (Liu B, et al (2016) Sci Rep 6:27352), LRA4 (L-KDR aldolase) promoter (Liu B, et al (2016) Sci Rep 6:27352), THI11 (Thiamine biogenic protein) promoter (Stadlmayr G, et al (2010) J Biotechnol 150:519-529), GTH1 (High affinity glucose transporter) promoter (Koller A, et al (2000) Yeast 16:651-656), CUP1 (Copper-binding metallothionein protein) (Landes N, et al (2016) Biotechnol Bioeng 113(12):2633-2643), PPLCC1 (Laccase) promoter (Landes N, et al (2016) Biotechnol Bioeng 113(12):2633-2643), GAP (Glyceraldehyde-3-phosphate dehydrogenase) promoter (Waterham HR, et al (1997) Gene 186:37-44), YPT1 (GTPase involved in secretion) promoter (Sears IB, et al (1998) Yeast 14:783-790), TEF1 (Translation elongation factor-1 alpha) promoter (Stadlmayr G, et al (2010) J Biotechnol 150:519-529), GCW14 (Glycosylphosphatidylinositol) promoter (Liang S, et al (2013) Biotechnol Lett 35 (11):1865-1871), and PGK1 (Phosphoglycerate kinase). In certain embodiments, the promoter included in the nucleic acid construct is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

[0100] Depending on the promoter used, the nucleic acid construct or expression vector as used herein can be termed as AOX1 promoter-, ADH3 promoter-, DAS promoter-, FLD1 promoter-, PEX8 promoter-, ICL1 promoter-, LRA3 promoter-, LRA4 promoter-, THI11 promoter-, GTH1 promoter-, CUP1 promoter-, PPLCC1 promoter-, GAP promoter-, YPT1 promoter-, TEF1 promoter- GCW14 promoter-, or PGK1 promoter-based nucleic acid construct or expression vector. In certain embodiments, the nucleic acid construct or expression vector as used herein is a GAP promoter-based nucleic acid construct or expression vector.

[0101] As used herein, the term "operably linked" refers to arrangement of fragments such that transcription is initiated by a promoter, and proceeds to the termination code through amino acid coding sequences.

[0102] In addition to the polynucleotide encoding the KlenTaq polymerase and a promoter operably linked thereto, the nucleic acid construct as used herein may include various elements known to be involved in helping the expression / secretion of KlenTaq polymerase.

[0103] In an embodiment, the nucleic acid construct herein further comprises a transcription terminator. The transcription terminator is essential for the processing and polyadenylation of messenger RNA. The transcription terminator includes those known to be used inP. pastoris, example of which includes AOX1 terminator.

[0104] In certain embodiments, the nucleic acid construct herein further comprises a nucleic acid sequence encoding a histidine tag, which is linked to the terminus of the polynucleotide encoding the KlenTaq polymerase. The histidine tag is used to facilitate protein purification using affinity chromatography. The addition of histidine to the terminus of the protein of interest leads to a significantly increased metal ion affinity of the protein, enabling easy purification. Contacting a protein having a histidine tag with a column onto which metal ions such as nickel are immobilized under conditions of pH 8.0 or higher allows the histidine tag to chelate the metal ions and bind to the column, thereby recovering the target protein with high purity.

[0105] The nucleic acid sequence encoding the histidine tag can be linked to the C-terminus or N-terminus of the polynucleotide encoding the KlenTaq polymerase. In certain embodiments, the nucleic acid sequence encoding the histidine tag is linked to the C-terminus of the polynucleotide encoding the KlenTaq polymerase.

[0106] The histidine tag may consist of at least 6 histidine residues. In one embodiment, the histidine tag consists of 6 histidine residues (hexahistidine). In another embodiment, the histidine tag consists of 7 histidine residues (heptahistidine). In another embodiment, the histidine tag consists of 8 histidine residues (octahistidine). In another embodiment, the histidine tag consists of 9 histidine residues (nonahistidine). In another embodiment, the histidine tag consists of 10 histidine residues (decahistidine). The number of histidine constituting the histidine tag is readily adjustable by those skilled in the art.

[0107] In one embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding a (GSG)nlinker between the polynucleotide encoding the KlenTaq polymerase and the nucleic acid sequence encoding the histidine tag, wherein n is an integer from 1 to 5, G denotes glycine, and S denotes serine.

[0108] The (GSG)nlinker allows for interaction between the polynucleotide encoding the KlenTaq polymerase and the nucleic acid sequence encoding the histidine tag or increases the spatial separation between the two domains. Direct fusion of the polynucleotide encoding the KlenTaq polymerase with the nucleic acid sequence encoding the histidine tag may cause undesirable results, such as misfolding of the fusion protein, low protein yield, or dysfunction.

[0109] It was found by the inventors that direct fusion of a histidine tag to KlenTaq polymerase leads to poor purification, because KlenTaq polymerase does not bind well to the column during purification (data not presented).

[0110] Although various linkers composed of glycine and serine, such as GS linkers, GSG linkers, GSSG linkers, GGGG linkers, and GGGGS linkers, are known in the art, it was found by the inventors that GSG linkers are the most effective in the expression and purification efficiency of KlenTaq polymerase.

[0111] The length of the (GSG)nlinker can be optimized by adjusting the copy number n. In certain embodiments, the copy number n in the (GSG)nlinker is 1. In certain embodiments, the copy number n in the (GSG)nlinker is 2.

[0112] In one embodiment, the nucleic acid construct used herein further comprises an intervening sequence between the polynucleotide encoding the KlenTaq polymerase and the nucleic acid sequence encoding the histidine tag. In certain embodiments, the intervening sequence includes a single or multiple cloning site, such as a restriction site. The intervening sequence is used for inserting the polynucleotide encoding the KlenTaq polymerase in front of the nucleic acid sequence encoding the histidine tag.

[0113] According to the present disclosure, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter and the polynucleotide encoding the KlenTaq polymerase.

[0114] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the KlenTaq polymerase, and a transcriptional terminator.

[0115] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the KlenTaq polymerase, the nucleic acid sequence coding the histidine tag, and a transcriptional terminator.

[0116] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the nucleic acid sequence encoding the histidine tag, the polynucleotide encoding the KlenTaq polymerase, and a transcriptional terminator.

[0117] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the KlenTaq polymerase, a nucleic acid sequence encoding a (GSG)nlinker, the nucleic acid sequence encoding the histidine tag, and a transcriptional terminator.

[0118] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the nucleic acid sequence encoding the histidine tag, a nucleic acid sequence encoding a (GSG)nlinker, the polynucleotide encoding the KlenTaq polymerase, and a transcription terminator.

[0119] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the KlenTaq polymerase, a nucleic acid sequence encoding a (GSG)nlinker, an intervening sequence, the nucleic acid sequence coding the histidine tag, and a transcription terminator.

[0120] In certain embodiments, the nucleic acid construct as used herein includes, from N-terminus to C-terminus, a promoter, the nucleic acid sequence encoding the histidine tag, an intervening sequence, a nucleic acid sequence coding a (GSG)nlinker, the polynucleotide encoding the KlenTaq polymerase, and a transcription terminator.

[0121] In certain embodiments, the expression vector comprises a plurality of the nucleic acid constructs. In certain embodiments, the plurality of the nucleic acid constructs are composed of 6 to 16 nucleic acid constructs. In certain embodiments, the plurality of the nucleic acid constructs are composed of 8 to 12 nucleic acid constructs. In certain embodiments, the plurality of the nucleic acid constructs are composed of 8 nucleic acid constructs. In certain embodiments, the plurality of the nucleic acid constructs are composed of 10 nucleic acid constructs. In certain embodiments, the plurality of the nucleic acid constructs are composed of 12 nucleic acid constructs.

[0122]

[0123] Expression vector

[0124] The expression vector as used herein includes the nucleic acid construct(s) described above.

[0125] As used herein, the term "expression vector" refers to a vehicle designed to express a protein, such as KlenTaq polymerase, in a cell, such as a cell ofP. pastoris.

[0126] The expression vector herein comprises a nucleic acid construct, which is fundamental to expression, and may also comprise various other components.

[0127] In certain embodiments, the expression vector further comprises an origin of replication.

[0128] In certain embodiments, the expression vector further comprises a selectable marker. The selectable marker may include a nucleic acid sequence encoding an antibiotic resistance gene. Examples of such antibiotic resistance genes include, but are not limited to, a bleomycin resistance gene, a phleomycin resistance gene, or a zeocin resistance gene.

[0129] Bleomycin and phleomycin are glycopeptide antibiotics of bleomycin family, isolated from a mutant strain ofStreptomyces verticillus. Phleomycin binds to DNA and is intercalated into it, destroying the integrity of the double helix. Phleomycin is known to be active against most bacteria, filamentous fungi, yeast, plant, and animal cells.

[0130] Zeocin is a member of bleomycin / phleomycin family of antibiotics and is known to show strong toxicity against bacteria, fungi (including yeast), and plants and mammalian cell lines (Calmels et al., 1991; Drocourt et al., 1990; Gatignol et al., 1987; Mulsant et al., 1988; Perez et al., 1989).

[0131] In one embodiment, the antibiotic resistance gene as described above is BleoR. In one embodiment, the BleoR is a gene encoding an antibiotic binding protein that imparts resistance to bleomycin, phleomycin, and zeocin, and specifically a Sh ble gene derived fromStreptoalloteichus hindustanus.

[0132] The expression vector of the present disclosure is characterized in that it lacks a polynucleotide encoding a secretion signal peptide. In certain embodiments, the secretion signal peptide is characterized in that it is derived from a yeast.

[0133] Typically, a secretion signal peptide plays a role in promoting the secretion of proteins expressed inside a host cell to the outside of the cell,i.e., into the medium, during the culture of the transformed cell. Several secretion signal peptides have been reported in the art, includingSaccharomyces cerevisiaeα-mating factor (Poirier, N. et al. J. Agric. Food Chem. 2012, 60, 9807-9814),Saccharomyces cerevisiaemodified α-mating factor (MAF) (Xiong, A.S. et al. J. Biochem. Mol. Biol. 2004, 37, 282-291), α-amylase (Paifer, E. et al. Yeast 1994, 10, 1415-1419), Exg1p (Liang, S. et al. Biotechnol. Lett. 2013, 35, 97-105), inulinase (Massahi, A. et al. J. Theor. Biol. 2015, 364, 179-188), lysozyme (Oka, C. et al. Biosci. Biotechnol. Biochem. 1999, 63, 1977-1983), serum albumin (Xiong, R. et al. Biotechnol. Appl. Biochem. 2008, 51, 129-134), invertase (Kuberl, A. et al. J. Biotechnol. 2011, 154, 312-320), proteinase K (Gunkel, F. A and Gassen H. G. Eur. J. Biochem. 1989, 179, 185-194), or aqualysin I (Oledzka, G, et al. Protein Expr. Purifi. 2003, 29, 223-229) to express heterologous proteins extracellularly inP. pastoris.

[0134] However, the present inventors have found that KlenTaq polymerase, upon expression using an expression vector containing a secretion signal peptide inP. pastoris, is not secreted extracellularly, but remains within the cell in a state bound to the secretion signal peptide. Instead, it has been found that the use of an expression vector lacking a secretion signal peptide can increase the expression of KlenTaq polymerase into cells ofP. pastoris. This is contrary to previous reports that it is effective to use expression vectors with secretion signal peptides for expression of target proteins inP. pastoris.

[0135] Without wishing to be bound by theory, this is probably because KlenTaq polymerase cannot be secreted out of the cell due to its large size, even with the help of secretion signal peptides, and the presence of secretion signal peptides has an adverse effect on the expression of KlenTaq polymerase.

[0136] Expression vectors lacking a secretion signal peptide may be prepared by various engineering techniques known in the art. As an example, expression vectors with a secretion signal peptide, such as pgApzαA vector (J. Wu et al. (2016); J. S. Lee et al. (2018); Sams et al. (2017)) can be digested with appropriate restriction enzymes, such asBstI andSalI, to remove only the secretion signal peptide contained therein, such as α-mating factor. As another example, a commercially available expression vector lacking a secretion signal peptide, such as pPICZ (Thermo Fisher Scientific) or pGAPZ (Thermo Fisher Scientific), can be directly used.

[0137] The expression vector according to the present disclosure may be one selected from various vectors known in the art to be applicable toP. pastoris. Examples include, without limitation, pPIC9K vector having His4, Kan, and Amp as selectable markers (Fu, Zhao, Xiong, Tian, and Peng (2011), Tu et al. (2013), X. Chen et al. (2012), Apte-Deshpnade, Mandal, Soorapaneni, Prasad, Kumar, and Padmanabhan (2009)); pPICZα vector having Sh ble as a selectable marker (Goodrick et al. (2001), Chan et al. (2018); Prabhu, Veeranki, and Dsilva (2016); J. Li et al. (2017); Baeshen et al. (2016); Bardiya and Chang (2017)); pHIL-S1 vector having His4 and Amp as selectable markers (Ben Azoun, Belhaj, Gongrich, Gasser, and Kallel (2016); Satomura, Kuroda, and Ueda (2015); Chahardooli, Niazi, Aram, and Sohrabi (2016)); pgApzαA vector having Sh ble as a selectable marker (J. Wu et al. (2016); J. S. Lee et al. (2018); Sams et al. (2017)); pJL-Sx vector having FLD1 and Amp as selectable markers (Sunga and Cregg (2004)), pBLHIS-SX vector having His4 and Amp as selectable markers (Li et al. (2010)), or variants thereof.

[0138] As described above, the expression vector as used herein may comprise a plurality of the nucleic acid constructs,i.e., one or more copies of the nucleic acid constructs. In certain embodiments, the expression vector comprises 6 to 16 nucleic acid constructs. In certain embodiments, the expression vector comprises 8 to 12 nucleic acid constructs. In certain embodiments, the expression vector comprises 8 nucleic acid constructs. In certain embodiments, the expression vector comprises 10 nucleic acid constructs. In certain embodiments, the expression vector comprises 12 nucleic acid construct.

[0139] To our knowledge, there are no known expression vectors containing 6 or more, particularly 8 or 12 nucleic acid constructs. On the other hand, the present inventors have found that expression vectors containing a multimer of nucleic acid constructs of considerable size are effective for high expression of target proteins, particularly KlenTaq polymerase.

[0140] The expression vector containing the plurality of the nucleic acid constructs may be prepared by any method known in the art.

[0141] As an example, an expression vector containing a single nucleic acid construct is digested by an appropriate restriction enzyme to remove the nucleic acid construct. Then, a plurality of the nucleic acid constructs are linked together to produce a multimer of a desired size. Subsequently, the multimer is incorporated into the expression vector to produce an expression vector containing a plurality of the nucleic acid constructs (a multimer of the nucleic acid constructs).

[0142] As a specific example, an expression vector containing a single nucleic acid construct comprising a GAP promoter, a polynucleotide encoding KlenTaq polymerase, a nucleic acid sequence coding a histidine tag, and a transcriptional terminator is digested by restriction enzymesBglII andBamHI to obtain the nucleic acid construct. Thereafter, the obtained nucleic acid construct is linked to the same nucleic acid construct. Since the sticky end generated byBglII digestion can be linked to the sticky end generated byBamHI digestion, the end ofBamHI-digested nucleic acid construct can be linked to the end ofBglII-digested nucleic acid construct. However, since linkage products between the sticky end generated byBglII digestion and the sticky end generated byBamHI digestion are not digested even byBglII orBamHI, it is possible to construct a multimer, such as a dimer.

[0143] An expression vector containing a desired number of nucleic acid constructs can be selected by treating host cells containing the expression vector with increasing concentrations of an antibiotic or by checking the sizes of the nucleic acid constructs from the host cells, for example, by electrophoresis.

[0144] As described above, the use of an expression vector containing a plurality of nucleic acid constructs can increase the copy number of an expressed target protein to improve its expression efficiency. According to the present disclosure, inclusion of a plurality of nucleic acid constructs can be referred to as increased copy number or multimerization of a gene encoding for a target protein.

[0145]

[0146] Pichia pastoris

[0147] According to the method of the present disclosure, the host cell used to produce KlenTaq polymerase is a strain ofP. pastoris.

[0148] TheP. pastorisstrain may be one of a variety of strains known to be capable of producing foreign proteins. Examples include, without limitation, CBS7435 (NRRL Y-11430) (Kuberl A et al., (2011) J Biotechnol 154:312-320), DSMZ 70382 (CBS 704) (Mattanovich D et al., (2009) Microb Cell Fact 8:29), X-33 (Life technologies), GS115 (Cregg JM et al., (1985) Mol Cell Biol 5:3376-3385), GS190, JC220, JC254 (Cregg JM et al., Methods Mol Biol 103:17-26), KM71 (Cregg JM et al., (1987) In: Biological Research on Industrial Yeasts (Stewart GG, Russell I, Klein RD and Hiebsch RR, Eds., Vol. 2, pp. 1-18. CRC Press, Boca Raton, FL.), KM71H (Life technologies), SMD1163, SMD1165, SMD1168 (Gleeson MA et al., (1998) Methods Mol Biol 103:81-94), kex1 (Boehm T et al., (1999) Yeast 15: 563-572; Ni Z et al., (2007) Yeast 25:1-8), kex2 (Werten MWT et al., (2005) Appl Environ Microbiol 71:2310-2317), ysp1 (Yao XQ et al., (2009) J Biotechnol 139:131-136; Werten MWT et al., (2005) Appl Environ Microbiol 71:2310-2317), GlycoSwitch-Gal2 (Jacobs PP et al., Nat Protoc 4:58-70), GlycoSwitch-Man5 (Jacobs PP et al., Microb Cell Fact 9:93), YSH597 (Hamilton SR et al., (2006) Science 313:1441-1443), YGLY4140 (Choi B-K et al., (2003) Proc Natl Acad Sci USA 100:5022-5027), and ku70 (Naatsaari L et al., (2012) PLoS One 7:e39720).

[0149] In certain embodiments, the strain ofP. pastorisfor the transformation isP. pastorisX-33 (Thermo Fisher Scientific).

[0150]

[0151] Transformation

[0152] According to the method of the present disclosure,P. pastorisis transformed with an expression vector having a nucleic acid construct which comprises a polynucleotide encoding the KlenTaq polymerase, and a promoter operably linked thereto.

[0153] The transformation ofP. pastoriscan be performed by any of various methods known in the art.

[0154] As used herein, the term "transformation" means that a foreign DNA is imported into host cells and integrated into host chromosomal DNA (homologous recombination). The transformation ofP. pastorisaccording to the present disclosure can be performed, for example, by electroporation, protoplasmic fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agrobacterium-mediated transformation, PEG (polyethylene glycol), dextran sulfate, lipofectamine, or particle bombardment.

[0155] The transformation according to the present disclosure induces homologous recombination between the expression vector and the genome ofP. pastoris.

[0156] The homologous recombination described above arises from the similarity of sequences between the expression vector and the genome ofP. pastoris.

[0157] In one embodiment, the homologous recombination is based on sequence similarity between a promoter found in the expression vector and a promoter within the genome ofP. pastoris. Examples of the promoters can be found elsewhere herein, including glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

[0158] In one embodiment, the method of the present disclosure further comprises linearizing the expression vector prior to the step (b),i.e., transformation.

[0159] Linearization of the expression vector enables the expression vector to be incorporated into the genomic DNA ofP. pastorisvia homologous recombination to generate stable transformants.

[0160] In one embodiment, linearization of the expression vector can be achieved by using a restriction site present in the expression vector.

[0161] In one embodiment, linearization of the expression vector can be achieved by using a restriction site upstream of the nucleic acid construct.

[0162] In certain embodiments, linearization of the expression vector can be achieved by using a restriction site upstream of the promoter in the nucleic acid construct.

[0163] In certain embodiments, linearization of the expression vector can be achieved by using a restriction site 3 to 150 bp, 5 to 100 bp, 5 to 50 bp, 5 to 40 bp, 5 to 30 bp, or 5 to 20 bp upstream from the promoter in the nucleic acid construct.

[0164] Examples of restriction enzymes for the restriction site include, but are not limited to,BglII orNsiI.

[0165]

[0166] The step (a) as described above allows transformants ofP. pastoristo be obtainable.

[0167] In one embodiment, the genome of the transformedP. pastorisin which the homologous recombination was induced, contains (promoter-polynucleotide encoding KlenTaq polymerase)mderived from the expression vector, where m is an integer of 1 to 16.

[0168]

[0169] Step (b): Culture of Transformed P. Pastoris

[0170] In this step, the transformedP. pastorisis cultured in a culture medium under conditions permitting intracellular expression of the KlenTaq polymerase.

[0171] The culture medium may be a conventional medium which contains a carbon source and a nitrogen source required for culturing yeast. In one embodiment, the culture medium does not contain methanol.

[0172] Examples of the culture medium include, but are not limited to, YPD (Yeast extract Peptone Dextrose) medium, YPDS (Yeast extract Peptone Dextrose Sorbitol) medium, MGY (Minimal Glycerol) medium, MGYH (Minimal Glycerol Histidine) medium, MD (Minimal Dextrose) medium, MDH (Minimal dextrose Histidine) medium, MM (Minimal Methanol) medium, MMH (Minimal Methanol Histidine) medium, BMGH (Buffered Minimal Glycerol) medium, BMMH (Buffered Minimal Methanol) medium, BMGY (Buffered Glycerol complex) medium, and BMMY (Buffered Methanol complex) medium.

[0173] The culture medium may further comprise an antibiotic to select transformants transformed with the expression vector comprising the polynucleotide encoding KlenTaq polymerase according to the present disclosure. The antibiotic is one corresponding to a selectable marker contained in the expression vector.

[0174] In certain embodiments, the culture medium according to the present disclosure is YPDS medium containing an antibiotic, such as zeocin.

[0175] The composition of the YPDS medium typically includes 1% yeast extract, 2% peptone, 2% dextrose (glucose), and 1 M sorbitol, but it will be understood by those skilled in the art that the composition can be adjusted as needed.

[0176] Such antibiotic, such as zeocin, may typically be contained in a culture medium in an amount of 100 μg / mL, but the amount is readily adjustable by those skilled in the art.

[0177] The conditions for culturingP. pastorismay typically be at 20 to 40℃, preferably at 30℃, for 1 to 10 days, particularly for 1 to 7 days, more particularly for 2 to 3 days, and most particularly for 20 to 30 hours.

[0178]

[0179] Step (c): Lysis of Cultured P. Pastoris and Purification of Intracellularly Expressed KlenTaq Polymerase

[0180] In this step, the culturedP. pastorisis lysed and intracellularly expressed KlenTaq polymerase is then purified from the lysate.

[0181] The lysis of the culturedP. pastorismay be performed using any of methods known in the art.

[0182] In one embodiment, the lysis of the culturedP. pastorisis performed mechanically after separating cells from the culture ofP. pastoris.

[0183] The mechanical cell lysis may include lysis by sonication, lysis by French press, and lysis by a bead mill.

[0184] Cell lysis by sonication refers to a process of using sound energy greater than 16 kHz to disrupt the cell wall and cell membrane. However, sonication has the disadvantage of denaturing heat-sensitive enzymes. Meanwhile, cell lysis by French press refers to a process of filling cells into a hollow stainless-steel cylinder, which is often used on a laboratory scale, then extruding the cells under high pressure to an atmospheric pressure state through a needle valve at the bottom of the cylinder. Also, cell lysis by a bead mill refers to a process of stirring beads made of glass or iron to disrupt cells with high shearing force and impact force.

[0185] In certain embodiments, the lysis of the culturedP. pastorisis achieved by a process comprising the steps of: (i) centrifuging a culture ofP. pastoristo obtain cell pellets, and (ii) adding glass beads to the obtained cell pellets to disrupt cells.

[0186] For example,P. pastorismay be lysed by adding about 1 to 3 times as much glass beads (e.g., commercially available from Merck) and a suitable buffer (e.g., Breaking buffer) to the cell pellets and repeatedly vortexing.

[0187] In another embodiment, the lysis of the culturedP. pastorisis performed non-mechanically. Examples include treating the culture ofP. pastoriswith an enzyme such as lysozyme to lyse cells or slowly freezing cells and then thawing them to lyse the cell membrane.

[0188]

[0189] The purification of the KlenTaq polymerase from the lysedP. pastoris(lysate) may be performed by any of chromatographic methods known in the art.

[0190] Examples of chromatographic methods that can be used include, but are not limited to, immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC), and reverse phase HPLC.

[0191] In one embodiment, the purification according to the present disclosure is performed using affinity chromatography for histidine protein. Examples of such affinity chromatography include, but are not limited to, Ni-NTA affinity chromatography (e.g., using Cytiva HisTrapTMFast Flow column).

[0192] In one embodiment, the purification according to the present disclosure is performed using anion exchange chromatography. Examples of such anion exchange chromatography include, but are not limited to, HiPrepTMQ FF or HiTrapTMQ FF.

[0193] In one embodiment, the purification according to the present disclosure is performed using primary purification by affinity chromatography, followed by secondary purification by anion exchange chromatography.

[0194]

[0195] The use of the method of the present disclosure enables obtaining KlenTaq polymerase expressed within the cells ofP. Pastoris.

[0196] According to the method of the present disclosure, the purified, intracellularly expressed KlenTaq polymerase is produced in an amount of 0.1 to 1.5 mg per gram of cells ofP. pastoris. In one embodiment, the purified, intracellularly expressed KlenTaq polymerase is produced in an amount of 1 to 15 mg per 100 mL of the culture medium. The production yield may be improved by optimizing the cultivation in step (b) and the lysis and purification in step (c) of the method of the present disclosure. Specifically, appropriate culture medium and culture conditions capable of achieving maximum expression ofP. pastoristransformed according to the present disclosure, and appropriate lysis and purification capable of achieving maximum yield of the expressed KlenTaq polymerase will allow the KlenTaq polymerase to be recovered in high yield.

[0197] Using an expression vector containing a secretion signal peptide leads to accumulation of KlenTaq polymerase bound to the secretion signal peptide within a cell without secreting KlenTaq polymerase outside the cell, whereas the method of the present disclosure accumulates KlenTaq polymerase without a secretion signal peptide within a cell. As described above, KlenTaq polymerase bound to a secretion signal peptide has no commercial value because of its structural instability, while the KlenTaq polymerase obtained by the method of the present disclosure is of high commercial value due to having no secretion signal peptide bound.

[0198] According to the method of the present disclosure, the purified, intracellularly expressed KlenTaq polymerase is free of nucleic acids derived fromE. coli. In other words, the purified, intracellularly expressed KlenTaq polymerase is not contaminated with nucleic acids derived fromE. coli.

[0199] Thus, the purified, intracellularly expressed KlenTaq polymerase does not lead to any false positives due to a non-targetE. coliin an amplification reaction. That is, an amplification reaction using the purified, intracellularly expressed KlenTaq polymerase does not result in false positives due toE. coli-derived non-target nucleic acids. For example, the purified, intracellularly expressed KlenTaq polymerase does not generate a signal at all or generates a signal having a Ct value of 40 or higher, in the absence of a target nucleic acid in a nucleic acid amplification reaction, such as a PCR reaction. A signal having a Ct value of 40 or higher is considered to be insignificant and thus to indicate the absence of the target nucleic acid in a sample.

[0200] According to our finding, a sample containing theuidA gene fromE. coliproduced an amplification product in an amplification reaction using the KlenTaq polymerase according to the present disclosure, whereas a sample not containing the gene (negative control) did not produce any amplification product in the amplification reaction (see Fig. 12).

[0201]

[0202] In an aspect, the present disclosure provides KlenTaq polymerase, prepared by the method as described above.

[0203] In one embodiment, the KlenTaq polymerase is free of nucleic acids derived fromE. coli.

[0204] In one embodiment, the KlenTaq polymerase does not lead to any false positives from a non-targetE. coliin an amplification reaction.

[0205] Since the detailed description of the KlenTaq polymerase is found elsewhere herein, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

[0206]

[0207] In an aspect, the present disclosure provides a method of amplifying a target nucleic acids in a sample using the KlenTaq polymerase as described above.

[0208] The amplification of the target nucleic acid may be performed by a nucleic acid amplification reaction known in the art, such as PCR or real-time PCR. The nucleic acid amplification reaction may include the steps of amplifying a target nucleic acid in a sample using primers and detecting the amplified target nucleic acid. The amplified target nucleic acid may be detected by measuring a fluorescence signal generated from a probe having a label, such as a fluorescent label, linked.

[0209] The method may be for detecting a target nucleic acid fromE. coli.

[0210] Since commercially available KlenTaq polymerases are typically produced by anE. coliexpression system, they are likely to be contaminated with DNA derived fromE. coli. Therefore, it is difficult to determine whether a positive result in a nucleic acid amplification reaction using such contaminated KlenTaq polymerase is due to the presence of targetE. colior the presence of contaminatedE. coli. On the other hand, since the KlenTaq polymerase obtained by the method of the present disclosure does not contain nucleic acids from contaminatedE. coli, positive results in the nucleic acid amplification reaction can be reliably translated into the presence of targetE. coli.

[0211]

[0212] In an aspect, the present disclosure provides a transformedPichia pastoris, prepared by transformingP. pastoriswith an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding a KlenTaq polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.

[0213] Since the details described above are described elsewhere herein, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

[0214]

[0215] The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative, and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

[0216]

[0217] Example 1: Intracellular expression of KlenTaq polymerase using expression vector not containing secretion signal peptide

[0218]

[0219] <1-1> Construction of expression vectors

[0220] For intracellular expression of KlenTaq polymerase inP. pastoris, an expression vector lacking a secretion signal peptide was constructed as follows.

[0221] First, the nucleotide sequence of the wild-typeTaqDNA polymerase was codon-optimized according to the codon usage ofP. pastoris(see Table 1). Among the codon-optimized nucleotide sequence of the wild-typeTaqDNA polymerase, the nucleotide sequence corresponding to KlenTaq DNA polymerase is shown in SEQ ID NO: 1.

[0222] Subsequently, the codon-optimized nucleotide sequence of the wild-type polymerase as a template was subjected to PCR using Klen TaqBstBI forward primers (5'-ACGTATTCGAACGATGGGCCTTTTACACGAATTTGGTTTGCTC-3'; SEQ ID NO: 2) to generate aBstBI restriction site (TTCGAA), a CG dinucleotide, an initiation codon (ATG), and a trinucleotide encoding Gly (GGC) at the N-terminus of KlenTaq DNA polymerase, and Taq GSGSalI reverse primers (5'-TGATGGTCGACGCCGCTGCCTTCTTTAGCTGACAACCAGTCTTCTC-3'; SEQ ID NO: 3) to generate a Gly-Ser-Gly linker and aSalI restriction site at the C-terminus of KlenTaq DNA polymerase. The PCR product consists of (BstBI restriction site)-(codon-optimized KlenTaq polymerase)-(GSG linker)-(SalI restriction site). Afterwards, the PCR product was digested withBstBI andSalI.

[0223] Then, the pGAPZαA vector (Thermo Fisher Scientific Inc.; see Fig. 1) containing the alpha-mating factor (αA) signal peptide (282 bp; 89 amino acids) ofSaccharomyces cerevisiaewas purchased and digested withBstBI andSalI to remove the alpha-mating factor signal peptide from the pGAPZαA vector.

[0224] Thereafter, the PCR product and the pgApzαA vector, both digested withBstBI andSalI, were ligated to construct the expression vector, "pGAPZ KlenTaq pol".

[0225] After transformingE. coliDH10B with the expression vector pGAPZ KlenTaq pol, the transformant was grown on a low-sodium LB agar medium containing zeocin. The expression vector was isolated from the grown transformants and sequenced by Macrogen Co., Ltd. to confirm the correct construction of the pGAPZ KlenTaq pol.

[0226] The resulting expression vector includes a nucleic acid construct containing, from N-terminus to C-terminus, (GAP promoter)-(polynucleotide encoding KlenTaq polymerase)-(GSG linker)-(histidine tag)-(AOX1 terminator).

[0227] Then, in order to construct expression vectors containing a dimer of the nucleic acid constructs, the expression vector pGAPZ KlenTaq pol (about 4.44 kb) containing a monomer of the nucleic acid construct was digested withBglII and then treated with an alkaline phosphatase to block self-ligation. Then, the pGAPZ KlenTaq pol was digested withBglII andBamHI to obtain a fragment (about 2.52 kb) containing the pGAP promoter-KlenTaq polymerase. TheBglII-digested product and theBglII- andBamHI-digested fragment were ligated with each other, and the ligation product was transformed intoEscherichia coliDH10B. The ligation products were then isolated from the transformants and digested withBglII andBamHI to select the expression vector "pGApz KlenTaq pol 2M" (about 6.94 kb) containing a dimer (about 5.04 kb) of pGAP promoter-KlenTaq polymerase by agarose gel electrophoresis.

[0228] Again, in order to construct expression vectors containing a tetramer of pGAP promoter-KlenTaq polymerase, aBglII-digested pGApz KlenTaq pol 2M was ligated with a fragment containing a dimer of pGAP promoter-KlenTaq polymerase, obtained by digesting pGApz KlenTaq pol 2M withBglII andBamHI. Then, the ligation product was transformed intoE. coliDH10B. The ligation products were then isolated from the transformants and digested withBglII andBamHI to select the expression vector "pGApz KlenTaq pol 4M" (about 12 kb) containing a tetramer (about 10.04 kb) of pGAP promoter-KlenTaq polymerase by agarose gel electrophoresis.

[0229] Afterwards, above process was repeated for the pGApz KlenTaq pol 4M to obtain the expression vector "pGApz KlenTaq pol 8M" (about 22 kb). The pGApz KlenTaq pol 8M contains an octamer of the pGAP promoter and the polynucleotide encoding KlenTaq polymerase as a target protein.

[0230] Again, above process was repeated for the pGApz KlenTaq pol 8M to obtain the expression vector "pGApz KlenTaq pol 12M" (about 32.1 kb). The pGApz KlenTaq pol 12M contains a dodecamer of the pGAP promoter and the polynucleotide encoding KlenTaq polymerase as a target protein.

[0231] To confirm the correct construction of the expression vectors, the expression vectors were each digested withBglII andBamHI, followed by electrophoresis.

[0232] The electrophoresis results are shown in Figs. 2 to 5.

[0233] Fig. 2 shows the electrophoresis results of pGAPZ KlenTaq pol 2M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second lane represents the result of pGAPZ KlenTaq pol for comparison; and the third to seventh lanes represent the results of pGAPZ KlenTaq pol 2M obtained from five transformants. In each lane, the upper band corresponds to a monomeric (about 2.52 kb) or dimeric (about 5.04 kb) KlenTaq polymerase, and the lower band corresponds to a backbone (about 1.9 kb) of the expression vector except for the monomeric or dimeric KlenTaq polymerase. The results show that pGAPZ KlenTaq pol 2M prepared by the method of the present disclosure contains dimeric KlenTaq polymerase.

[0234] Fig. 3 shows the electrophoresis results of pGAPZ KlenTaq pol 4M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second lane represents the result of pGAPZ KlenTaq pol 2M for comparison; and the third to seventh lanes represent the results of pGAPZ KlenTaq pol 4M obtained from five transformants. In each lane, the upper band corresponds to a dimeric (about 5.04 kb) or tetrameric (about 10.08 kb) KlenTaq polymerase, and the lower band corresponds to a backbone (about 1.9 kb) of the expression vector except for the dimeric or tetrameric KlenTaq polymerase. The results show that pGAPZ KlenTaq pol 4M prepared by the method of the present disclosure contains tetrameric KlenTaq polymerase.

[0235] Fig. 4 shows the electrophoresis results of pGAPZ KlenTaq pol 8M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second lane represents the result of pGAPZ KlenTaq pol 4M for comparison; and the third to seventh lanes represent the results of pGAPZ KlenTaq pol 8M obtained from five transformants. In each lane, the upper band corresponds to a tetrameric (about 10.08 kb) or octameric (about 20.16 kb) KlenTaq polymerase, and the lower band corresponds to a backbone (about 1.9 kb) of the expression vector except for the tetrameric or octameric KlenTaq polymerase. The results show that pGAPZ KlenTaq pol 8M prepared by the method of the present disclosure contains octameric KlenTaq polymerase.

[0236] Fig. 5 shows the electrophoresis results of pGAPZ KlenTaq pol 12M digested byBglII / BamHI. The first lane represents a DNA ladder marker; the second and third lanes represent the results of pGAPZ KlenTaq pol 12M obtained from two transformants; and the fourth lane represents the result of pGAPZ KlenTaq pol 8M for comparison. In each lane, the upper band corresponds to an octameric (about 20.16 kb) or dodecameric (about 30.2 kb) KlenTaq polymerase, and the lower band corresponds to a backbone (about 1.9 kb) of the expression vector except for the octameric or dodecameric KlenTaq polymerase. The results show that pGAPZ KlenTaq pol 12M prepared by the method of the present disclosure contains dodecameric KlenTaq polymerase.

[0237]

[0238] <1-2> Obtaining transformants ofP. pastoris

[0239] The expression vectors, pGAPZ KlenTaq pol 8M and pGAPZ KlenTaq pol 12M, constructed in Example 1-1 were each linearized by digesting them withBglII and were transformed intoP. pastorisX-33.

[0240] Specifically,Pichia pastorisstrains were cultured overnight at 30℃ in 5 mL of YPD medium in a 50-ml conical tube, and then 0.1-0.5 mL of the culture was inoculated into 500 mL of fresh medium and grown overnight to OD600of 1.3-1.5. The cells were then centrifuged at 3000 Х g for 10 min at 4℃ to obtain cell pellets. 20 mL of transfection buffer (1 M LiAC 2 ml, 1 M DTT 0.2 ml, 1 M sorbitol 12 ml, 1 M Tris-HCl (pH 7.6) 0.2 ml, sterile water 5.6 mL) was added to the cell pellets, transferred to a 50-ml conical tube, and left to stand at room temperature for 30 min. Afterwards, the liquid was centrifuged at 3000 Х g for 7 minutes at 4℃ to obtain cell pellets. Again, 5-10 mL of 1 M sorbitol was added, suspended, and centrifuged. This process was repeated twice to completely remove the salt component in the medium. Afterwards, the cell pellets were suspended in 1 mL of cold 1 M sorbitol.

[0241] The cell suspension (80 μL) was mixed with the linearized expression vector (1-10 μg) which was dissolved in 5-10 μL of sterile distilled water afterBglII digestion and removal of salt components, and transferred to a cold electroporation cuvette. The cells were subjected to an electric pulse of 1500 V-2400 V, 1 mL of cold 1 M sorbitol was added, and then transferred to a sterile 50 mL tube. The tube was incubated at 30℃ without stirring for 1 h. Then, 1 mL of YPD medium was added to the tube, and was cultured at 30℃ with shaking at 200 rpm for 1 h. Approximately 200 μL of the culture was plated on YPDS medium containing 100 μg / mL zeocin, and incubated at 30℃ for 2-3 days to obtain formed colonies.

[0242]

[0243] <1-3> Intracellular expression of KlenTaq polymerase in transformedP. pastoris

[0244] It was investigated whether the transformedP. pastorisobtained in Example 1-2 expresses KlenTaq polymerase intracellularly.

[0245] Specifically, each of the transformants obtained in Example 1-2 was inoculated into 3 mL YPD medium containing 3 μL of zeocin (100 mg / mL) and cultured for 48 hours while stirring at 250 rpm at 30℃. The culture was then centrifuged to obtain cell pellets. The cell pellets were added with about twice glass beads (Merck G9268), 50 μL of breaking buffer (50 mM sodium phosphate pH 7.4, 1 mM PMSF, 1 mM EDTA, and 5% glycerol), and about 2 μL PMSF (phenylmethanesulfonylfluoride, 100 mM). The mixture was vortexed at maximum speed for 30 seconds, then cooled on ice for 1 minute, which was repeated 5 times. Then, another 50 μL of breaking buffer was added, vortexed, and heated at 80℃ for 30 minutes. After centrifugation, 16 μL of supernatant and 4 μL of dye were mixed and loaded onto SDS-PAGE.

[0246] SDS-PAGE results for cell lysates of each transformant are shown in Figs. 6 and 7.

[0247] In Fig. 6, the first lane represents a marker; the second lane represents the result for cell lysates of untransformedPichia pastorisX-33 for comparison, and the third to tenth lanes represent the results for cell lysates ofPichia pastorisX-33 transformed with pGAPZ KlenTaq pol 8M.

[0248] As shown in Fig. 6, untransformedPichia pastorisX-33 did not express KlenTaq polymerase intracellularly, whereas the transformants 8-12 and 8-14 expressed large amounts of KlenTaq polymerase intracellularly.

[0249] Meanwhile, in Fig. 7, the first lane represents a marker; the second to tenth lanes represent the results for cell lysates ofPichia pastorisX-33 transformed with pGAPZ KlenTaq pol 12M.

[0250] As shown in Fig. 7, the transformants 12-11 and 12-12 expressed large amounts of KlenTaq polymerase intracellularly.

[0251]

[0252] Example 2: Expression level of KlenTaq polymerase in Pichia pastoris transformed with expression vector containing KlenTaq polymerase

[0253] The expression level of KlenTaq polymerase according to the culture time by the transformant shown to express KlenTaq polymerase in Example 1 was examined.

[0254] Specifically,Pichia pastoris8-12 and 8-14 transformed with pGAPZ KlenTaq pol 8M, andPichia pastoris12-11 and 12-12 transformed with pGAPZ KlenTaq pol 12M were each inoculated into 3 mL YPD medium containing 3 μL of zeocin (100 mg / mL) and cultured for 24 hours at 30℃ with stirring at 250 rpm. Thereafter, 1% of the culture was inoculated into 30 mL YPD and cultured for 48 hours.

[0255] The growth ofPichia pastoris8-12, 8-14, 12-11, and 12-12 at 24, 28.5, 31.5, and 48 hours of incubation was compared by measuring the absorbance at 600 nm (OD600) of the cultures of the transformants. The results are shown in Table 2 below.

[0256] Time (hr)Transformant8-12Transformant8-14Transformant12-11Transformant12-12000002422.522.4523.2519.8528.526.327.1528.125.3531.53132.13131.654834.433.931.931.9

[0257] As shown in Table 2,Pichia pastorisobtained by the method of the present disclosure showed rapid growth until 31.5 hours, but showed a decrease in growth thereafter.

[0258] On the other hand, each culture taken at 24 hours, 28.5 hours, 31.5 hours, and 48 hours of the incubation was adjusted to an OD600of 10, and then centrifuged to obtain cell pellets.

[0259] Then, the cell pellets were added with about twice glass beads (Merck G9268), 50 μL of breaking buffer (50 mM sodium phosphate pH 7.4, 1 mM PMSF, 1 mM EDTA, and 5% glycerol), and about 2 μL PMSF (phenylmethanesulfonylfluoride, 100 mM). The mixture was vortexed at maximum speed for 30 seconds, then cooled on ice for 1 minute, which was repeated 5 times. Then, another 50 μL of breaking buffer was added, vortexed, and heated at 80℃ for 20 minutes. After centrifugation, 16 μL of supernatant and 4 μL of dye was mixed and loaded onto SDS-PAGE.

[0260] The results are shown in Figs. 8 and 9.

[0261] As seen in Figs. 8 and 9, it was found that the four transformants expressed a significant amount of KlenTaq polymerase during the culture. In particular, transformant 12-12 showed the highest expression level at 24 to 28.5 hours of incubation.

[0262]

[0263] Example 3: Production and purification of KlenTaq polymerase

[0264] The transformant 12-12 showing the highest expression level in Example 2 was inoculated into 6 mL YPD medium containing 6 μL of zeocin (100 mg / mL) and cultured at 30℃ (250 rpm) for about 24 hours.

[0265] Then, 2 mL of the culture was inoculated into 200 mL YPD medium containing 100 μL of zeocin and cultured at 30℃ (250 rpm) for about 24 hours. After culture, the final OD600was approximately 24.75 / mL.

[0266] The culture was aliquoted in an amount of about 100 mL per tube and the aliquots were each centrifuged to obtain about 10.2 g of cell pellets per 200 mL of the culture.

[0267] About 10.2 g of the cell pellets were mixed with 20 mL of breaking buffer (20 mM sodium phosphate pH 8.0, 20 mM KCl, 1 mM PMSF). Then, 5 mL of the mixture was dispensed into 50 mL conical tube containing 5 g of glass bead. The mixture was vortexed for 40 seconds, and then cooled in ice water for 2 minutes, which was repeated 10 times.

[0268] After the mixture was centrifuged at 9000 rpm for 20 minutes, the supernatant was collected in a separate tube and heated at 80℃ for 20 minutes. The pellet obtained by the centrifugation was mixed with 1 mL of breaking buffer containing 5 mM imidazole, vortexed for 40 seconds, and then cooled in ice water for 2 minutes, which was repeated 5 times. Afterwards, the mixture was heated at 80℃ for 20 minutes. The heated mixture was centrifuged to obtain the supernatant.

[0269] Through the above process, approximately 24.17 mL of cell lysate could be obtained. It was found that the amount of KlenTaq polymerase in the cell lysate was approximately 12.7 mg by the Bradford assay.

[0270] Afterwards, the cell lysate was purified by affinity chromatography using HisTrapTMFF column (5 mL). The purification conditions were as follows.

[0271] Flow rate (sample loading): 1 mL / min

[0272] Flow rate: 5 mL / min

[0273] Gradient: B 100% for 20 CV

[0274] Buffer: 20 mM phosphate buffer (pH 8)

[0275] Buffer A: 20 mM KCl; imidazole 5 mM

[0276] Buffer B: 20 mM KCl; imidazole 500 mM

[0277] The amount of the purified KlenTaq polymerase was determined by absorbance at 280 nm using an Epoch microplate spectrophotometer. As a result, it was confirmed that approximately 7.04 mg of protein was obtained.

[0278] Afterwards, the purified protein was purified again by anion exchange chromatography using HiTrapTMQ FF (5 mL). The purification conditions were as follows.

[0279] Flow rate: 5 mL / min

[0280] Gradient: B 100% for 25 CV

[0281] Buffer: 20 mM phosphate buffer (pH 8)

[0282] Buffer A: 20 mM KCl

[0283] Buffer B: 1 M KCl

[0284] The amount of the purified KlenTaq polymerase was determined by absorbance at 280 nm using an Epoch microplate spectrophotometer. As a result, it was confirmed that about 3.94 mg of protein was obtained.

[0285] Thereafter, the purified protein was concentrated by ultrafiltration to obtain about 2.1 mg of KlenTaq polymerase.

[0286] The results demonstrate that a large amount of KlenTaq polymerase can be obtained fromPichia pastorisaccording to the method of the present disclosure.

[0287]

[0288] Example 4: Polymerization activity of KlenTaq polymerase

[0289] It was examined whether the KlenTaq polymerase purified from cell lysates of the transformedP. pastorisin Example 3 had polymerization activity.

[0290] Specifically, the KlenTaq polymerase obtained in Example 3 was subjected to PCR. The PCR reaction was performed by mixing 2 mM dNTPs 2 μL, λ DNA 0.8 μL, 2 kb λ forward primer (SEQ ID NO: 4) 0.5 μL, 2 kb λ reverse primer (SEQ ID NO: 5), 2 μL of 10X Taq buffer, 1.0 μg / μL of the KlenTaq polymerase obtained in Example 3 and 13.2 μL of sterile water and subjecting it to 3 minutes at 94℃ and 30 cycles of 30 seconds at 95℃, 20 seconds at 58℃ and 20 seconds at 72℃. For comparison, 0.5 μg or 1.0 μg of wild-typeTaqpolymerase was also subjected to the same PCR reaction.

[0291] The PCR products were electrophoresed to confirm whether the λ DNA was normally amplified.

[0292] The electrophoresis results are shown in Fig. 10. The first lane represents a DNA ladder marker, the second lane represents the result of PCR products using 1 μg of KlenTaq polymerase obtained in Example 3, the third lane represents the result of PCR products using 0.5 μg of wild-typeTaqpolymerase, and the fourth lane represents the result of PCR products using 1 μg of wild-typeTaqpolymerase.

[0293] As shown in Fig. 10, it was found that the KlenTaq polymerase obtained by the method of the present disclosure amplified target nucleic acids in the PCR reaction comparable to wild-typeTaqpolymerase. Thus, it was confirmed that the KlenTaq polymerase according to the present disclosure has normal polymerization activity.

[0294]

[0295] Example 5: Specific activity of KlenTaq polymerase

[0296] The specific activity of the KlenTaq polymerase according to the present disclosure was determined.

[0297] Specifically, the EvaEZTMreagent (containing EvaGreen® dye, primed template, dNTPs, MgCl2, and Tris buffer), 3.125 ng, 1.563 ng, 0.781 ng, or 0 ng of the KlenTaq polymerase according to the present disclosure, and ultrapure water were added in a volume ratio of 10:1:9 (EvaEZ reagent : KlenTaq polymerase : ultrapure water) to 10X ThermoPol reaction buffer (New England Biolabs). The mixtures were placed into 8-strip PCR tubes in an amount of 20 μL each, and fluorescence was measured at 30-second intervals for 60 minutes at 72℃ using CFX96 Real-Time PCR thermocycler (Bio-Rad) to observe the kinetics. The data showing the initial linear function among the obtained data was used to calculate the slope. The slope (the x-axis represents the concentration of KlenTaq polymerase, and the y-axis represents derivative of time fluorescence value) is an indicator of dNTP consumption. The dNTP consumption measured by the slope is shown in Figs. 11A and 11B. One unit of KlenTaq polymerase was defined as the consumption of 4.29 nmole dNTP in 30 minutes.

[0298] As shown in Figs. 11A and 11B, it was found that the KlenTaq polymerase according to the present disclosure consumed 674.39 nmole dNTP per 1 μg, and therefore had a specific activity of 157.10 U / μg.

[0299]

[0300] Example 6: Amplification ofE. coligene using KlenTaq polymerase

[0301] PCR methods for detectingE. colitypically have used primers specific foruidA gene encoding β-D-glucuronidase (Bej A. K. et al. Appl. Environ. Microbiol. 1991, 307-14; Molina, F, et al. BMC Biotechnology 2015, 48).

[0302] In this Example, it was examined whether the KlenTaq polymerase obtained according to the method of the present disclosure can amplify the nucleic acid of targetE. coliwithout contamination of non-targetE. coligenomic DNA.

[0303] The PCR reaction was performed by mixing 2 μL of 2 mM dNTPs, 0.8 μL ofE. coligDNA, 0.5 μL each of primers specific foruidA gene (SEQ ID NOs: 6 and 7), 2 μL of 10X Taq buffer, 1 / 2 dilution (1.35 μg / μL), 1 / 5 dilution (0.54 μg / μL), or 1 / 10 dilution (0.27 μg / μL) of the KlenTaq polymerase obtained in Example 3, and 13.2 μL of sterile water and subjecting it to 3 minutes at 95℃ and 35 cycles of 30 seconds at 95℃, 20 seconds at 58℃ and 20 seconds at 72℃.

[0304] Then, the PCR products were electrophoresed on a 1.5% agarose gel.

[0305] The results are shown in Fig. 12. The first lane represents a DNA ladder marker; the second lane represents the result of PCR using a 1 / 2 dilution of KlenTaq polymerase according to the present disclosure in the presence ofE. coligenomic DNA; the third lane represents the result of PCR using a 1 / 2 dilution of KlenTaq polymerase according to the present disclosure in the absence ofE. coligenomic DNA; the fourth lane represents the result of PCR using a 1 / 5 dilution of KlenTaq polymerase according to the present disclosure in the presence ofE. coligenomic DNA; the fifth lane represents the result of PCR using a 1 / 5 dilution of KlenTaq polymerase according to the present disclosure in the absence ofE. coligenomic DNA; the sixth lane represents the result of PCR using a 1 / 10 dilution of KlenTaq polymerase according to the present disclosure in the presence ofE. coligenomic DNA; and the seventh lane represents the result of PCR using a 1 / 10 dilution of KlenTaq polymerase according to the present disclosure in the absence ofE. coligenomic DNA.

[0306] As shown in Fig. 12, the amplification product (162 bp) ofuidA gene was clearly visible in the samples containingE. coligenomic DNA, but not in the samples not containingE. coligenomic DNA. Additionally, the KlenTaq polymerase according to the present disclosure amplified theuidA gene ofE. colieven at low amounts.

[0307] The results demonstrate that the KlenTaq polymerase according to the present disclosure can amplify the targetE. coligene without contamination of any non-targetE. coligenomic DNA.

[0308]

[0309] Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

1.A method for preparing KlenTaq polymerase lacking 5' to 3' exonuclease activity, comprising:(a) transformingPichia pastoriswith an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the KlenTaq polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide;(b) culturing the transformedP. pastorisin a culture medium under conditions permitting intracellular expression of the KlenTaq polymerase; and(c) lysing the culturedP. pastorisand purifying the intracellularly expressed KlenTaq polymerase from the lysate.2.The method of claim 1, wherein the polynucleotide encoding the KlenTaq polymerase is codon-optimized forP. pastoris.3.The method of claim 2, wherein the polynucleotide encoding the KlenTaq polymerase comprises a sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto.4.The method of claim 1, wherein the promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.5.The method of claim 1, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a histidine tag, which is linked to the terminus of the polynucleotide encoding the KlenTaq polymerase.6.The method of claim 5, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a (GSG)nlinker between the polynucleotide encoding the KlenTaq polymerase and the nucleic acid sequence encoding the histidine tag, wherein n is an integer from 1 to 5, G denotes glycine, and S denotes serine.7.The method of claim 1, wherein the expression vector further comprises an antibiotic resistance gene as a selectable marker.8.The method of claim 7, wherein the antibiotic resistance gene is a bleomycin resistance gene, a phleomycin resistance gene, or a zeocin resistance gene.9.The method of claim 1, wherein the expression vector comprises a plurality of the nucleic acid constructs.10.The method of claim 9, wherein the plurality of the nucleic acid constructs are composed of 6 to 16 nucleic acid constructs.11.The method of claim 1, further comprising linearizing the expression vector prior to the transformation of step (a).12.The method of claim 1, wherein the transformation of step (a) induces homologous recombination between the expression vector and the genome ofP. pastoris.13.The method of claim 1, wherein the purified, intracellularly expressed KlenTaq polymerase is produced in an amount of 0.1 to 1.5 mg per gram of cells ofP. pastoris.14.The method of claim 1, wherein the purified, intracellularly expressed KlenTaq polymerase is free of nucleic acids derived fromE. coli.15.The method of claim 1, wherein the purified, intracellularly expressed KlenTaq polymerase does not lead to any false positives from a non-targetE. coliin an amplification reaction.16.KlenTaq polymerase, prepared by the method of any one of claims 1 to 15.17.The KlenTaq polymerase of claim 16, which is free of nucleic acids derived fromE. coli.18.The KlenTaq polymerase of claim 17, which does not lead to any false positives from a non-targetE. coliin an amplification reaction.19.A method of amplifying a target nucleic acid in a sample using the KlenTaq polymerase of claim 16.20.The method of claim 19, wherein the target nucleic acid is fromE. coli.21.A transformedPichia pastoris, prepared by transformingP. pastoriswith an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding a KlenTaq polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.

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