PROCESS FOR PRODUCING pPIC9K-KEX2 RECOMBINANT PLASMIDS IN ESCHERICHIA COLI BACTERIAL CELLS FOR CO-EXPRESSION IN THE YEAST PICHIA PASTORIS

Abstract

The process for producing pPIC9K-KEX2 recombinant plasmids in E. coli bacterial cells for co-expression in the yeast Pichia pastoris. This invention aims to provide the process for producing pPIC9K-KEX2 recombinant plasmids using the bacteria E. coli to increase the expression level of human insulin glargine (hIG) in yeast Pichia pastoris. According to this invention, the following are the process stages for producing the recombinant plasmids: creating a primer design for the ORF isolation process using the PCR method and for the KEX2 ORF isolation from the yeast P. pastoris GS115 strain. The sequence for the pair of primers used is KEX2-Fw-BamHI:5'GGG GGG GAT CCA TGT ATT TGC CAG CAC TT-3 ' and KEX2-REV-EcoRI: 5' GGG GGG AAT TCT TAC AAT GCC GCA CGT TT - 3'. Next, pPIC9K-KEX2 P. pastoris is constructed with the following stages: using kanamycin as an antibiotic to select the target recombinant bacteria and inserting the KEX-2 gene of Pichia pastoris into the pPIC9K plasmid at the unique sites of BamHI and EcoRI. With this construction, the insert will be in frame with the α-factor signal peptide under the control of the AOX1 promoter. The above process will result in recombinant plasmids containing the KEX2 ORF.

Description

[0001] Description

[0002] PROCESS FOR PRODUCING pPIC9K-KEX2 RECOMBINANT PLASMIDS IN

[0003] ESCHERICHIA COLI BACTERIAL CELLS FOR CO-EXPRESSION IN THE YEAST

[0004] PICHIA PASTORIS

[0005] Technical Field of the Invention

[0006] This invention relates to a process for producing pPIC9K~ KEX2 recombinant plasmids in E. coli bacterial cells , more speci fically, the process of producing ppic9k-KEX2 recombinant plasmids in E. coli bacterial cells for co-expression in the yeast plasmids .

[0007] Background of the Invention

[0008] Site-speci fic proteolysis is a common mechani sm in the protein maturation process and plays an important role in activating many enzymes and peptide hormones . In the secretory pathway of eukaryotic cells , this mechanism is mainly mediated by kexin-like proteases (KEX2 ) , a sub- family of serine subtilisin-like proteinases ( Bader et al . , 2008 ) . In nature, KEX2 is an intracellular protease found in the endoplasmic reticulum and trans-Golgi membrane (Mizuno et al . , 1988 ) .

[0009] KEX-2 is a protease with a molecular weight (BM) of ~ 67 KDa .

[0010] It is produced by yeast and has speci fic cleavage activity at the amino acid sequence -Lys-Arg -or -Arg-Arg KEX-2 plays a role in processing the precursors from yeast and has a critical role in yeast secretion pathways by removing peptide signals from pre-proteins and releasing the mature form of the secreted protein . In recent years , protease enzymes have been commonly used to puri fy recombinant proteins . For example, such enzymes are used for separating target proteins from fusing with nontarget proteins , such as protein tagging . However, the utili zation of KEX-2 for recombinant protein puri fication is still not as popular as other types of proteases , such as enterokinase and carboxypeptidase A and B .

[0011] KEX-2 is also found in P. pastori s (Newport Q5J881 ) . It is in the same family as S 8 and has a similarity of 46 . 42 % with KEX-2 in S. cerevisiae. The role of this protease in both yeasts is to process the cx-mating factor, cleave peptide signals from pre-proteins, and mature protein secretion (Mizuno et al., 1988; Manfredi et al., 2016) . KEX-2 has been reported and implemented to improve the downstream maturation process of the insulin glargine protein through co-expression in the yeast Pichia pastoris . This is done by making two recombinant plasmid constructs. Both will be introduced into Pichia pastoris , which secrete the precursor insulin glargine. The recombinant plasmids can be constructed by fusing the KEX2 gene in frame with the constitutive promoter , glyceraldehyde-3-p hosphate dehydrogenase (GAP) . Meanwhile, the other construct is fused with the inducible promoter formaldehyde dehydrogenase (FLD1) . Each of these constructs is subcloned into the pPICZA expression vector placed between the sites Xhol and EcoRI (Sreenivas et al., 2014; Waterham et al., 1997; Duan et al., 2009) . The pPICZA vector is a shuttle vector that enables intracellular gene expression. Both results snow that HIG is active with a greater percentage than without the introduction of recombinant KEX-2.

[0012] The journal publication by Sreenivas et al. (2015) titled "Enhancement in the production of recombinant two-chain insulin glargine by the over-expression of KEX-2 protease in Pichia pastoris, " mentions the process of producing recombinant plasmid pPICZA-KEX2) . The target gene is a synthetic coding sequence that codes for the yeast KEX2 Saccharomyces cerevisiae. pPICZA is a plasmid / expression vector that can express proteins intracellularly. Intracellularly expressed proteins require a longer purification stage than proteins expressed extracellular. Pratiwi et al. also produced a recombinant plasmid KEX-2 (pD902- KEX2) from the yeast S. cerevisiae and the bacteria E. coll. However, they added the His tag linker to help with the purification stage.

[0013] A patent published in 2019 by researchers from Fujian Normal University with the number (CN110438022A) has also made a PGABZB-KEX-2 recombinant plasmid in the bacteria E. coll TOP10 Strain to be expressed on P. pastoris cells. PGABZ is an expression vector for P. pastoris and is designed for the constitutive expression of proteins. The pair of primers used are KEX2-up and KEX-2 down, which adds an EcoRI enzyme restriction site to each primer. The primers and PCR processes used differ from those employed by the inventor of this claim.

[0014] Another patent with the number EPO327377A2 has described their success in producing several recombinant plasmids that carry the endoprotease KEX2 using the recombinant DNA technique in the bacterial cells E. coll DH-1, two of which are pYE-KEX2A3 and pYE~kexA4. The promoter they used is constitutive. KEX-2 Endoprotease is inserted into plasmids with different constructs, namely KEX-2 containing hydrophobic regions and soluble KEX2 without hydrophobic regions. The KEX2 open reading frame (ORF) is isolated from the yeast chromosome of Saccharomyces cerevisiae using the Cryer et al. method.

[0015] Patent number US8936917B2 entitled "KEX-2 Cleavage Regions of Recombinant Fusion Proteins", aims to improve the cleavage process of proteins that will be expressed in fungal cells by fusing the recombinant plasmid pTrex4-her 2. There are 3 KEX-2 DNA sequences in its construction: the KEX-2 region, the KEX-2 site, and the KEX-2 site pre-sequence.

[0016] Unlike the previous inventions, the pPIC9K plasmid used in this invention is a shuttle vector, which is made to secrete proteins extracellularly. This makes the purification stage of proteins secreted into the medium easier. In this invention, KEX-2 is constructed to be expressed in the yeast P. pastoris extracellularly .

[0017] This construction aims to allow two approaches to increase the active HIG protein, i.e., in situ and in the laboratory. For in situ, we measure the active HIG obtained from the KEX-2 reaction secreted by P. pastoris and HIG secretion in the same P. pastoris, which KEX-2 will activate the secreted HIG in situ.

[0018] Summary of the Invention

[0019] This invention aims to provide the process for producing the pPIC9k-KEX2 recombinant plasmid in the bacteria E. col achieve this goal, in this invention, the KEX2 gene is isolated from the P. pastoris gsll5 strain using the PCR technique with a pair of specially designed primers. In addition, the inventor optimised the PCR conditions for this invention. The production process begins with conducting a PCR reaction of the KEX2 gene isolated from P. pastoris with a pair of specially designed primers and following PCR reaction stages with optimized conditions. Before transformation into bacterial cells, the KEX2 gene and the pPIC9K plasmid are fused together by a ligase reaction using the T4 DNA ligase enzyme. The next stage is to introduce the results of the ligase reaction into the bacterial cell E. coll Dh5-α strain using the heat-shock transformation method. Transformant selection was performed by growing bacteria on LB media containing the kanamycin antibiotic at 37oC. Molecular weight analysis using 1% agarose gel obtained a ppik9k-KEX2 recombinant plasmid with a size of 2350 bp.

[0020] Brief Description of the Figures

[0021] Figure 1 shows the results of the ORF isolation by the PCR method described in this invention.

[0022] Figure 2 shows the temperature optimization results for the primary attachment described in this invention.

[0023] Figure 3 shows the double digest results of the plasmids pPIC9K and KEX2 ORF using the restriction enzymes BamHI and EcoRI, as described in this invention.

[0024] Figure 4 shows the results of the confirmation and target recombinant screening by the colony PCR technique, as described in this invention.

[0025] Figure 5 shows the confirmation results of the recombinant plasmid using the Sal I restriction enzyme analysis, as described in this invention.

[0026] Description of the Invention

[0027] This invention describes the process for producing plasmids that can be co-expressed with HIG inserted into P. Pastoris . In the laboratory, KEX-2 and HIG are mixed according to several ratios and compared to find the best ratio for increasing active

[0028] HIG . The recombinant plasmids produced in this invention will be used for single or simultaneous secretions with HIG in P pastoris . In addition, the selection process will be easier as the plasmids will be produced using pPIC9K with kanamycin as the selectable marker and will be co-expressed in a recombinant P. pastori s that will express human insulin glargine . This is because the antibiotics used for the selection process of recombinant P. pastoris are different , namely the antibiotics zeocin and geneticin .

[0029] This invention relates to the production process for a recombinant plasmid pPIC9K-KEX2 in the bacteria E. coli . The production of recombinant plasmids consists of the following steps :

[0030] 1 . Creating a primer design that will be used in the isolation process .

[0031] 2 . The following aspects should be considered for the primer selection process ; the primer ' s secondary structure should not have a hairpin loop structure, the percentage of GC base content in the primer dimer should be considered, and the addition of restriction enzymes should be adj usted to the unique si tes in plasmids with the condition that the restriction enzyme does not cut the KEX-2 Pi chia area as the cloning target . To obtain the full-length of the KEX-2

[0032] Pichia pastori s. primer design should be done in the upstream region containing the ATG start codon . The DNA sequence for the forward primer is KEX2-Fw-BamHI : 5-GGG GGG

[0033] GAT CCA TGT ATT TGC CAG CAC TT-3 ' . We also introduced the unique restriction site for the enzyme BamHI and the stop codon

[0034] TAA in the downstream region . The DNA sequence for the reverse primer is KEX2-REV-EcoRI : 5 ' GGG GGG AAT TGT TAG AAT GGG GCA CGT

[0035] TT-3r) . We also introduced the unique restriction site for the

[0036] EcoRI enzyme . The DNA primer sequence above was chosen because it does not form a secondary structure, ensuring that the PGR process runs eff iciently. Meanwhile , unique sites were introduced to each primer because we will insert a full length KtX-2 Pichia pastoris gene to the pPIC9K plasmid in frame with the alpha Factor signal peptide .

[0037] 3. Isolation of KEX2 ORF from, the yeast P. pastoris cells . To obtain the KEX2 ORF from yeast, the first step is the total genomic isolation of the yeast P. pastoris . This isolation is conducted using the Yea.Star G e n o m i c DNA kit equipped with the zymo lyase enzyme, which plays a. role in breaking down the yeast cell wall at a specific temperature. The result of this isolation is the total DNA of the yeast P. pastori s . This total DNA will be used as a PCR reaction template . The results of the genomic DNA obtained will undergo the PCR method with the KEX2-Fw-BamHI and KEX2-REV-

[0038] ECoRI primers . The PCR reaction conditions used were predenaturation at 94 °C for 2 minutes , denaturation at 98 f o r 30 seconds , annealing at 55°C for 30 seconds , and extension at OS^C for 3 minutes. The last three conditions were conducted for 35 cycles . Molecular- weight analysis using the DNA electrophoresis method was performed to analyse the PCR results.

[0039] 4 The vector pPIC9K was used to construct a recombinant plasmid inserted with ORF isolated from the P. pastoris genome . The

[0040] ORF obtained from the vector was cut using- the BamHI- EcoRI restriction enzymes . The recombinant vector inserted with the KEX2 ORF was transformed into competent E. coli strain

[0041] DH5a cells . The competent cells were then added with the ligation results. The transformation was conducted by the heat shock method. The trans formant colonies that grew were further re-cultivated on solid LB media containing the antibiotic kanamycin at 100 μg / mL. The recombinant plasmids were then isolated using a plasmid isolation kit, and the results were characterised by their molecular weight using restriction enzymes (Figure 3) . The confirmation was also conducted using the PCR method (Figure 4) The selection process for the pPIC9k-KEX2 recombinant plasmid produced only uses kanamycin . The selection process for this recombinant plasmid is expected to be easier when it is coexpressed in P. pastoris recombinants that express human insulin glargine . This is because the antibiotics used for the selection process on P. pastori s recombinants are different, namely using the antibiotics zeocin and geneticin . Such a construction will result in plasmids with an insert criterion that, is in frame with the cx-factor signal peptide under the control of the A0X1 promoter .

Claims

ClaimsA process for producing pPIC9K-KEX2 recombinant plasmids in Escherichia coii bacterial cells for co-expression in the yeast Pichia pastoris, which consists of: a. designing primers to obtain the primer designs for KEX2- Fw-BamHI and KEX2~REV~EcoRI while considering the following aspects : i. the secondary structure of the primers should not form a hairpin loop structure; ii. the percentage of GC bases in primer dimers should be considered; iii. the addition of restriction enzymes is tailored to the unique site in the plasmid, but it must not cut the KEX-2 Pichia region that is the cloning target; b. isolating KEX2 open reading frames (ORF) from P. pastoris yeast cells, which includes the following stages: i. isolating the total genome of the yeast P. pastoris using the YeaStar'TMGenomic DNA kit equipped with the enzyme zymolyase plays a role in breaking down the yeast cell wall at a specific temperature. The total DNA of the yeast P. pastoris will be obtained from this isolation process. This total DNA will be used as a PCR reaction template ; ii. conducting PCR using KEX2-Fw-BamHI and KEX2-REV - EcoRI primers with the following PCR reaction conditions : predenaturation at 94oC for 2 minutes, denaturation at 98oC for 30 seconds, annealing at 55oC f foorr 320 m siencuotnedss,, denaturation at and extension a 68oC fCor fo 3r0 s 3ec moinndust,es a. The last three conditions are performed for a total of 35 cycles; iii. obtaining KEX2 isolates (ORF) ; c. performing ORF construction on the pPIC9K vector by combining the KEX2 gene and pPIC9K plasmid through a ligase reaction using the T4 DNA ligase enzyme; d. transforming the stage (c) results into E. coli cells; e. selecting target recombinant bacteria by growing E. collbacteria on LB media containing 50 pg / ml of kanamycin at a temperature of 370C and confirming the recombinant plasmid i solation results with 1 % agarose gel ; and f . obtaining pPIK9k-KEX2 recombinant plasmids with a si ze of 2350 bp .The recombinant plasmids pPIC9K-KEX2 in the Escherichia coli bacteria for co-expression in the yeast Pichia pastoris consist of a pair of primers with the following nucleotide base sequence :® KEX2~Fw~BamHI : 5 ' -GGG GGG GAT CCA TGT ATT TGC CAG CAC TT~ 3 ’ ; and• KEX2-REV-EcoRI : 5 ' GGG GGG AAT TCT TAG AAT GCC GCA CGT TT-

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