Patent Application
20230365978
The present application claims priority to Korean Patent Application No. 10-2022-0059486, filed on May 16, 2022. The entire contents of the above-listed application are hereby incorporated by reference for all purposes.
The material in the accompanying sequence listing submitted electronically in XML format is hereby incorporated by reference in its entirety into this application. The accompanying file, named PLS22320_sequence_listing_ST26.XML was created on Nov. 30, 2022 and is 223 KB.
The following disclosure relates to a recombinant vector containing a hybrid signal sequence and a method for secretory production of a human insulin-like growth factor-1 (hIGF1) using the same, and more particularly, to a method of producing a hIGF1 that may implement improvement of secretory expression of hIGF1 by fusing, to the N-terminus of hIGF1, a hybrid signal sequence obtained by fusing a signal sequence of hydrophobin gene Srh1 of Trichoderma harzianum with a pro-sequence of an α-mating factor of yeast (Saccharomyces cerevisiae) and expressing hIGF1.
An insulin-like growth factor-1 (IGF1), also called somatomedin C, is mainly produced and secreted by the liver and also by most tissues without any specific restrictions. The IGF1 produced through stimulation of growth hormone secreted by the pituitary gland plays an important role in regulating cell proliferation, differentiation, and apoptosis through induction of tyrosine phosphorylation by stimulating IGF1 receptors on surfaces of various types of cells of muscle, bone, nerve, ovary, kidney, and the like through mechanisms such as endocrine, paracrine, and autocrine mechanisms.
Accordingly, the IGF1 has availability not only as a material for treating diseases such as growth deficiency, peripheral nerve regeneration, Type 1 diabetes, rheumatoid arthritis, and osteoporosis but also as a functional material for cosmetics, and thus, various attempts have been made for mass production thereof.
However, hIGF1 having a structure similar to that of insulin is a single-chain protein consisting of 70 amino acids and having a size of about 7 kDa and has three disulfide bonds. For these reasons, there is a problem in that it is difficult to express the IGF1 as an active form having a proper three-dimensional structure in Escherichia coli (E. coli), which is a prokaryotic host widely used for production of a useful recombinant protein. Recently, it has been reported that the above problem is alleviated by using E. coli Shuffle T7 as a host that has an improved disulfide bond-forming ability in the cytoplasm by fusing disulfide bond oxidoreductase (DsbA) to the amino terminus of IGF1 and inhibiting the activity of thioredoxin reductase (trxB) and glutathione reductase (gor) (Emamipour N, Vossouqhi M, Mahboudi F, Golkar M, Fard-Esfahani P, Applied Microbiology and Biotechnology, 103, 3393-3406 (2019)). In addition to such a disulfide bond-forming ability, when a medical protein such as an IGF1 is a target protein, the typical endotoxin of E. coli, which is a gram-negative bacterium, may be another problem.
A unicellular eukaryotic host such as yeast, for example, Saccharomyces cerevisiae or Komagataella phaffii (reclassified name for Phichia pastoris) is attracting attention as a host for producing various medical proteins because it has an endoplasmic reticulum, which is an organelle that may support proper disulfide bonds, is endotoxin-free, and generally recognized as a safe (GRAS) organism that may be relatively rapidly grown and cultured at a high concentration in a minimal medium. In these yeasts, foreign proteins are mainly produced by a secretory expression method in which a signal sequence is fused to an amino terminus of a target protein and secreted into the medium. The secretory expression efficiency varies greatly depending on the type of signal sequence and the target protein fused therewith, whereas the standard for compatibility between the protein and the signal sequence is not clearly established. Therefore, it may be important to discover various types of secretion signal sequences. In particular, in the case of hIGF1, it has been reported that only an α-mating factor among known signal sequences may be used to induce secretory expression (U.S. Patent Publication No. 5,521,086). Therefore, there is an urgent need to discover various signal sequences or develop a rational design method for secretory expression and production of a hIGF1.
(Patent Document 1) Korean Patent Laid-Open Publication No. 2014-0146163
(Patent Document 2) U.S. Patent Publication No. 5,521,086
An embodiment of the present invention is directed to providing a method of producing a hIGF1 that may implement significant improvement of secretory expression of the hIGF1 in Komagataella phaffii by producing a hybrid signal sequence synthesized by fusion of heterologous secretion signal sequences.
In one general aspect, there is provided a hybrid signal sequence consisting of an amino acid sequence for secretory expression of a hIGF11 (hIGF1) and obtained by fusing a pro-sequence of an α-mating factor to the C-terminus of a signal sequence of hydrophobin Srh1 containing one or two or more amino acid mutations.
The amino acid mutation may be obtained by inserting arginine into an amino acid at position 2 of the N-terminus of the signal sequence of the hydrophobin Srh1.
The hydrophobin Srh1 may be derived from Trichoderma harzianum, and the α-mating factor may be derived from yeast (Saccharomyces cerevisiae).
In another general aspect, there is provided a polynucleotide encoding the hybrid signal sequence.
The resulting polynucleotide encoding hybrid signal sequence may consist of a base sequence of SEQ ID NO: 5.
In still another general aspect, a recombinant expression vector contains the polynucleotide encoding hybrid signal sequence and a hIGF1.
The resulting polynucleotides may be repeated 2 to 10 times, thus present the corresponding copy in chromosome.
The recombinant expression vector may consist of a base sequence of SEQ ID NO: 6.
In still another general aspect, there is provided a non-human transformant transformed by introducing the recombinant expression vector into a chromosome.
The transformant may be Komagataella phaffii.
In still another general aspect, a method of producing a recombinant protein includes: preparing the recombinant expression vector; transforming the recombinant expression vector into a non-human transformable host to obtain a transformant; culturing the transformant to overexpress a hIGF1; and purifying and recovering an overexpressed recombinant protein.
In still another general aspect, there is provided a recombinant protein produced by the method of producing a recombinant protein.
The recombinant protein may be a recombinant protein from which a hybrid signal sequence is completely removed.
Other features and aspects will be apparent from the following detailed description, the drawings, and the claims.
Hereinafter, a production method and use of a hIGF1 based on a hybrid signal sequence of the present invention will be described in detail with reference to the accompanying tables and drawings.
The drawings described herein are provided by way of example so that the spirit of the present invention can be sufficiently transferred to those skilled in the art. Therefore, the present invention is not limited to the drawings and may be implemented in other forms. In addition, the drawings will be exaggerated in order to provide the spirit of the present invention.
Terms “first”, “second”, and the like may be used to describe various elements, but the elements should not be limited by the terms. The terms are only used to distinguish one element from another element. For example, a first element may be named a second element and the second element may also be similarly named the first element without departing from the scope of the present invention.
Technical terms and scientific terms used herein have the general meanings understood by those skilled in the art to which the present invention pertains unless otherwise defined, and a description for the known function and configuration unnecessarily obscuring the gist of the present invention will be omitted in the following description and the accompanying drawings. Terms defined in commonly used dictionaries should be interpreted as having meanings that are consistent with their meanings in the context of the related art, and will not be interpreted as having ideal or excessively formal meanings unless otherwise clearly defined in the present specification.
In addition, unless the context clearly indicates otherwise, singular forms used in the specification of the present invention may be intended to include plural forms.
In addition, a unit used in the specification of the present invention without special mention is based on weight, and as an example, a unit of % or a ratio refers to wt % or a weight ratio.
In addition, the expression “comprise(s)” used in the specification of the present invention is intended to be an open-ended transitional phrase having an equivalent meaning as “include(s)”, “contain(s)”, “have (has)”, or “is (are) characterized by” and does not exclude an element, a material, or a step that is not further listed. In addition, the expression “consist(s) essentially of” means that a specific element, material, or step that is not listed in combination with another element, material, or step may be present in an amount having no unacceptably significant influence on at least one basic and novel technical idea of the present invention. In addition, the expression “consist(s) of” means the presence of only the listed element, material, or step.
The terms “component”, “composition”, “composition of compound”, “compound”, “drug”, “pharmacologically active agent”, “active agent”, “cure”, “therapy”, “treatment”, and “medicine” are used interchangeably to refer to a compound or compounds or a composition of a substance that induces a desired pharmacological and/or physiologic effect by a local and/or systemic action when administered to a subject (human or animal).
The terms “treatment” and “therapy” (as well as different forms thereof) used in the specification of the present invention include preventative (for example, prophylactic), curative, or palliative treatments. The term “treating” used herein includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease, or disorder.
The term “sample” or “specimen” used in the present invention refers to a subject for analysis, and is used in the same meaning throughout the specification.
The term “vector”, “expression vector”, or “recombinant expression vector” used in the specification of the present invention is a linear or circular DNA molecule that includes an element and an additional fragment provided for gene transcription and translation, and encodes a polynucleotide operably linked thereto. The additional fragment includes a promoter, a transcription termination sequence, and the like. The vector, the expression vector, or the recombinant expression vector includes one or more replication origins, one or more selection markers, and the like. The vector, the expression vector, or the recombinant expression vector is generally derived from a plasmid or viral DNA or contains both elements. The vector, the expression vector, or the recombinant expression vector may be linearized and recombined to be introduced into a chromosome of a host, if necessary.
The term “recombinant protein” used in the specification of the present invention refers to a common expression protein that expresses a gene derived from a cell of another species using a heterologous host and includes a protein in which another protein is linked to an amino or carboxyl terminus of a target protein sequence, or another amino acid sequence is added, if necessary.
An aspect of the present invention relates to a hybrid signal sequence consisting of an amino acid sequence for secretory expression of a hIGF1 and obtained by fusing a pro-sequence of an α-mating factor to the C-terminus of a signal sequence of hydrophobin Srh1 containing one or two or more amino acid mutations.
The hIGF1 has three pairs of disulfide bonds although it is a single-chain protein having a small size. The disulfide bond plays an important role in formation of a proper three-dimensional structure having a function and is one of the main causes of insoluble expression in Escherichia coli (E. coli), which is a prokaryotic organism having a reductive cytoplasm in which the proper formations of disulfide bonds are difficult, and the like. In addition to the disulfide bond-forming ability, when a medical protein such as an IGF1 is the target protein, endotoxin of E. coli, which is a gram-negative bacterium, may be another problem. Therefore, it is important for the hIGF1 to be designed to induce functional secretory expression as a recombinant protein in a eukaryotic cell that has an endoplasmic reticulum, which is an organelle that may form proper disulfide bonds and is classified as an endotoxin-free generally recognized as safe (GRAS) organism, instead of in a prokaryote cell.
Hydrophobin is a 10 to 15-kDa protein secreted by mycelial fungi, which are eukaryotic cells, and has four disulfide bonds. The exact pathway by which the hydrophobin is secreted by fungi is not well documented.
The amino acid mutation may mean, but is not limited to, substitution, addition, deletion, and the like of an amino acid. As a specific example of the present invention, the amino acid mutation may mean that arginine is inserted into an amino acid at position 2 of the N-terminus of the signal sequence of hydrophobin Srh1. The amino acid at position 2 of the N-terminus is known to affect the N-end rule that determines a localization, folding landscape, and interaction with chaperones (thus, protein fate) of a normally expressed protein.
The signal sequence of the hydrophobin Srh1 may be derived from, but is not limited to, different species. As a specific example of the present invention, the signal sequence of the hydrophobin Srh1 may be derived from Trichoderma harzianum. The signal sequence of the α-mating factor may also be derived from, but is not limited to, different species, which may be different from that of the hydrophobin. As a specific example of the present invention, the signal sequence of the α-mating factor may be derived from yeast (Saccharomyces cerevisiae).
Another aspect of the present invention relates to a polynucleotide encoding the hybrid signal sequence.
The polynucleotide is not particularly limited as long as it is a base sequence encoding the hybrid signal sequence. As an example, the polynucleotide may consist of a base sequence of SEQ ID NO: 5. In a process of producing the hybrid signal sequence, in order to improve transcriptional efficiency, the polynucleotide may introduce a kozak sequence and also randomly substitute a synonymous codon for a codon of the base sequence encoding about 10 or more amino acids at the N-terminus. For efficient transcription and translation of hIGF1, when a kozak sequence is designed, a process such as a rational design may be introduced so that a base that appears with a high frequency in a known kozak sequence may be located at a position other than the adenine (A) position that appears with the highest frequency, but the present invention is not limited thereto.
Still another aspect of the present invention relates to a recombinant expression vector containing the polynucleotide and a polynucleotide encoding a hIGF1. The recombinant expression vector may consist of, but is not limited to, a base sequence of SEQ ID NO: 6.
The recombinant expression vector may use a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, or the like, as a template, but the present invention is not limited thereto. As a preferred example, the recombinant expression vector may optionally contain expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, and may be prepared in various forms depending on a purpose.
In order to screen a transformant into which the recombinant expression vector is introduced, the recombinant expression vector may contain an antibiotic resistance marker, and the antibiotic resistance marker may be internal to the vector or may be externally introduced, but the present invention is not limited thereto.
The polynucleotides may be repeated 2 to 10 times in a host chromosome, but are not limited thereto. An expression level of the protein may be efficiently increased by increasing the copy number of the gene through the above process, which is preferable.
Still another aspect relates to a non-human transformant transformed with the recombinant expression vector.
The transformant may be used without limitation except for a human transformant as long as it may efficiently secrete and produce a recombinant protein. As a specific example of the present invention, Komagataella phaffii, which is a eukaryote, may be used, and a hIGF1 may be effectively secreted and produced using the transformant, which is preferable.
When the transformant is prepared, a transformation may be performed by a conventional method known in the art, and may be performed by, for example, a method of introducing a vector into a host by a natural introduction method, a thermal shock method, an electric shock method, or the like, and re-introducing the vector into a chromosome by homologous recombination, but the present invention is not limited thereto.
Still another aspect relates to a method of producing a recombinant protein, the method including: preparing the recombinant expression vector; transforming the recombinant expression vector into a non-human transformable host to obtain a transformant; culturing the transformant to overexpress a hIGF1; and purifying and recovering an overexpressed recombinant protein.
In culturing of the transformant, culture conditions are not particularly limited, and known culture conditions may be introduced and used. As a specific example, the culture may be performed at 28 to 32° C., preferably 29 to 31° C., and more preferably 30° C., and soluble expression of 90% or more of a target protein may be implemented in the above culture temperature range. A medium for culturing microorganisms may also be appropriately introduced from known media. As a specific example, a buffered methanol complex (BMMY) (100 mM potassium phosphate, pH 5.4, 1% yeast extract, 2% Bacto peptone, 1.34% YNB, and 400 μg/L biotin) medium may be used, but the present invention is not limited thereto.
The method of producing a recombinant protein may be performed by isolating and purifying a protein variant from a culture of the transformant. In this case, the culture may be a transformant or a culture medium thereof, and the culture medium may be a medium containing the transformant or a medium obtained by isolating the transformant.
Still another aspect relates to a recombinant protein produced by the method of producing a recombinant protein. The recombinant protein is not limited and may be an hIGF1 from which a hybrid signal sequence is completely removed through secretory expression and purification processes.
Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. Examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by Examples.
In the present invention, reagents, materials, and protocols used for a polymerase chain reaction (PCR), DNA cloning, transformation, and the like are as follows, which will be apparent to those skilled in the art.
Other reagents were purchased from Sigma-Aldrich (USA) and the like.
For secretory expression of a hIGF1, usability of each of signal sequences of nine types of hydrophobins derived from seven species was evaluated using SignalP.
The signal sequences of the hydrophobins are summarized in Table 1.
Ophiostoma
novo-ulmi
Trichoderma
reesei
Trichoderma
reesei
Trichoderma
viride
Pleurotus
ostreatus
Cladosporium
fulvum
Aspergillus
nidulans FGSC
Endothia
parasitica
Magnaporthe
grisea
The hydrophobicity of each of the signal sequences of the nine types of the hydrophobins shown in Table 1 were analyzed and compared with a signal sequence of yeast (Saccharomyces cerevisiae) Ost1 in which signal recognition particle (SRP)-dependent secretory expression was confirmed.
The results are illustrated in
When the signal sequences of the SRP-dependent Ost1 and the α-mating factor were compared with the signal sequence of the SRP-independent Pdip, it was confirmed that both types of the signal sequences had a hydrophobic core, but the changes in hydropathy thereof were significantly different (also see
Based on the assumption from this result that the factors determining the efficiency and SRP dependence of the signal sequence may be affected by the change in hydropathy, four types of CU, Hcf5, Mhp1, and Srh1 were selected as candidates and used in the following Examples, the CU and Hcf5 were predicted to be SRP-independent signal sequences due to a small change in hydropathy of the amino acid of the signal sequence, and the Mhp1 and Srh1 were predicted to be SRP-dependent signal sequences due to a large change in hydropathy.
Based on the report that the basic amino acid located at the N-terminus of the signal sequence is closely related to the post-translational translocation efficiency of exocrine proteins from the cytoplasm to the endoplasmic reticulum, a change in hydropathy of each of the signal sequences selected in Example 1 was analyzed again by inserting hydrophilic amino acid arginine (R) between amino acids at positions 2 and 3 of the N-terminus of the signal sequence of the hydrophobin having a relatively small change in hydropathy.
The results are illustrated in
It was confirmed from this that the change in hydropathy of the signal sequence was increased by inserting the hydrophilic amino acid into the N-terminus, and it was also confirmed through SignalP analysis that the signal sequence was predicted in the same region even after the amino acid was inserted.
It was confirmed that, after the insertion of hydrophilic amino acid arginine, in the case of the signal sequence predicted to be SRP-dependent, the value indicating the probability of functioning as a signal sequence and the value predicting the probability of cleavage for exocytosis were increased in the results obtained using SignalP, whereas in the case of Cu and Mhp1 predicted to be SRP-independent, all of the above values were decreased.
In order to improve the extracellular secretion efficiency of the hIGF1, a hybrid signal sequence was designed by fusing a pro-sequence of an α-mating factor to the C-terminus of the signal sequence of the hydrophobin modified through redesign in Example 2.
In order to prepare a vector in which the hybrid signal sequence and the hIGF1 were cloned, a pair of primers and restriction enzymes shown in Table 2 were used.
In order to generate the hybrid signal sequence designed by the above process, a polymerase chain reaction (PCR) was performed with pGAPzα (Invitrogen, ThermoFisher Scientific (Waltham, MA, USA)) as a template using a combination of CU(R)_α, HCF5(R)_α, MHP1(R)_α, and Srh1(R)1_α F forward primers and α_IGF1 R reverse primer, thereby obtaining a pro-sequence of an α-mating factor containing a portion of the C-terminal sequence of each signal sequence of hydrophobin.
Subsequently, a gene encoding IGF1 without the innate signal sequence from pET22a-IGF1 provided by the Korea Institute of Ocean Science and Technology (KIOST) was amplified using α_IGF1 F and α_IGF1 R, and a gene in which a portion of the C-terminal sequence of the signal sequence of the hydrophobin, the pro-sequence of the α-mating factor, and the hIGF1 were fused was obtained through overlapping PCR of the obtained two DNA fragments.
A polymerase chain reaction (PCR) was performed with partial δCU-αpro-IGF1, δHCF5-αpro-IGF1, δMHP1-αpro-IGF1, and δSrh1-αpro-IGF1 (here, δ means to contain only the C-terminus of each signal sequence of hydrophobin) obtained through the above process as templates using a combination of CU(R)_α, HCF5(R)_α, MHP1(R)_α, and Srh1(R)1_α F forward primers and IGF1 R reverse primer. Through this, a whole gene fragment in which a hybrid signal sequence obtained by fusing an intact signal sequence of hydrophobin with a pro-sequence of an α-mating factor (αpro) was fused with hIGF1.
Subsequently, the gene was cleaved with EcoRI and SphI restriction enzymes, ligation with pPinK-HC (Thermo Fisher Scientific (Waltham, MA, USA)) cleaved with the same enzymes was performed, and cloning was performed in E. coli XL1-Blue.
From the E. coli XL1-Blue, the four types of the prepared plasmids pPinkHC-CU(R)_αpro-hIGF1, pPinkHC-HCF(R)5_αpro-hIGF1, pPinkHC-MHP(R)1_αpro-hIGF1 and pPinkHC-Srh1(R)1_αpro-hIGF1 (see SEQ ID NOS: 1 to 4 and
Each of the plasmids linearized through the treatment with the restriction enzyme SpeI in 3.2 of Example 3 was introduced into the chromosome of Komagataella phaffii by electroporation, and the transformant was plated on a minimal dextrose (MD) medium (1.34% yeast nitrogen base (YNB) without amino acids, 2% dextrose, and 1.5% Bacto agar).
The cells were cultured at 30° C. for 3 to 5 days and the three colonies to reach a size of 1.5 to 2.0 mm first were selected, the selected colonies were plated on the same medium again, the cells were cultured a second time at 30° C. for 3 days, the grown colonies were inoculated into a BMG (100 mM potassium phosphate buffer, pH 5.4, 1.34% YNB, 2% glycerol) medium, and then, culture was performed at 30° C. and 240 rpm for 2 days.
Thereafter, the cells cultured in the BMG medium were inoculated into a BMMY (100 mM potassium phosphate, pH 5.4, 1% yeast extract, 2% Bacto peptone, 1.34% YNB, 1% methanol) liquid medium so that an absorbance (OD600) was 0.5, and the cells were cultured under the same conditions. The cells were cultured for 72 hours while adding 1% methanol at intervals of 24 hours, the supernatant was separated when 48 hours and 72 hours have elapsed, and the total protein present in the separated supernatant was concentrated 10 times by trichloroacetic acid (TCA) precipitation. The hIGF1 present in the prepared concentrate was subjected to 15% SDS-PAGE analysis (see
In order to evaluate the difference in secretory expression of the hIGF1 by limitation to the difference in hybrid signal sequence, an expression level was normalized by the copy number of genes introduced into the chromosome of Komagataella phaffii. The genome of the recombinant Komagataella phaffii was extracted with a YeaStar Genomic DNA kit (Zymo Research (Irvine, CA)), and a real-time quantitative polymerase chain reaction (qPCR) was performed by repeating 40 cycles of a process performed at 98° C. for 2 minutes and a process performed at 98° C. for 30 seconds, 58° C. for 30 seconds, and 72° C. for 30 seconds using a pair of CU(R)_α, HCF5(R)_α, MHP1(R)_α, and Srh1(R)1_α F forward and α_IGF1 R reverse primers using an AirMax real-time PCR system (Agilent). The genome of Komagataella phaffii having one AOX1 promoter into which no gene was introduced and a pair of AOX1 F (SEQ ID NO: 31) and AOX1 R (SEQ ID NO: 32) primers were used as control groups. The qPCR results were normalized using a gene encoding glyceraldehyde 3 phosphate dehydrogenase (GAPDH) commonly used as an internal standard, and the measured gene copy numbers were compared.
As a result, when the number of inserted genes and the results of SDS-PAGE and ELISA (IGF1 ELISA kit, Enzo Life Sciences, Inc. (Farmingdale, NY)) were comprehensively analyzed, it was confirmed that among the four hybrid signal sequences, Srh1(R)1_α(pro) and CU(R)_α(pro) were more useful for secretory production of the hIGF1 in comparison to the other two types.
The secretory expression of the hIGF1 by each of the two types of the selected hybrid signal sequences was reconfirmed using an ELISA kit. The results thereof are illustrated in
Through the results of analyzing the copy number by the method in 4.1 of Example 4, it was confirmed that the reason for the difference in productivity in spite of the same hybrid signal sequence was due to the number of genes inserted into the chromosome of Komagataella phaffii.
Therefore, hybrid Srh1(R)1_αpro was finally selected as a signal sequence for secretory expression of the hIGF1.
It may be confirmed in
The prepared library was cultured at 30° C. and the recombinant Komagataella phaffii was selected based on a colony phenotype without growth retardation. The results obtained by normalizing the secretory expression level of hIGF1 by the copy number through the processes 4.1 and 4.2 of Example 4 are illustrated in
From the chromosome of Komagataella phaffii into which the scvSrh1(R)1_αpro #8 hybrid signal sequence showing the highest productivity was inserted with reference to
From this, the base sequence of scvSrh(R)1-αpro was obtained through analysis of the base sequence by DNA sequencing (see SEQ ID NO: 5 and
Hereinafter, an additional experiment was carried out using the resulting hybrid signal sequence.
It was confirmed that the productivity of hIGF1 was increased by about 1.2 times through the synonymous codon substitution of the above Example. As in Example 4, in the case where the copy number of genes encoding a target protein was high in the chromosome of Komagataella phaffii, the effect of increasing the productivity of the foreign protein was confirmed.
However, in a case where a single gene was introduced into a recombinant vector as in the above method, it was difficult to control the copy number of genes introduced into the chromosome to increase. Therefore, in order to solve this problem, a method of simultaneously introducing several genes into a chromosome by repeating cloning of the same gene into one recombinant vector was applied. That is, the scvSrh(R)1-αpro base sequence selected in Example 4 was repeatedly introduced several times to prepare a plasmid in which the scvSrh(R)1-αpro hybrid signal sequences along with hIGF1 were repeated 6 times (see SEQ ID NO: 6 and
As a control group, an α-IGF1 (see SEQ ID NO: 7 and
The pair of primers for preparing the plasmid are as shown in Table 3.
The entire signal sequence of the α-mating factor for producing pPink-HC-α-hIGF1 was amplified through a polymerase chain reaction using the pGAPzα plasmid as a template and αF (SEQ ID NO: 40) and α_IGF1 R (SEQ ID NO: 41) as primers, and a hIGF1 containing a portion of the C-terminal sequence of the signal sequence of α-mating factor was amplified using α_IGF1 F (SEQ ID NO: 42) and IGF1 R(SEQ ID NO: 43).
The two genes were fused by overlapping PCR, and an α-hIGF1 gene fused with the signal sequence of the α-mating factor was obtained through PCR amplification using αF (SEQ ID NO: 40) and IGF1 R (SEQ ID NO: 43).
The obtained genes were treated with restriction enzymes EcoRI and SphI, and the treated genes were cloned into a pPinkHC vector treated with the same restriction enzymes, thereby producing pPinkHC_α-IGF1.
Thereafter, a polymerase chain reaction was performed with pPinkHC-scvSrh1(R)1-αpro-hIGF1 #8 and pPink-HC_α-hIGF1 as templates using In multi X F and In multi X R primers to amplifyan AOX promoter-scvSrh1(R)1-αpro-hIGF1/αIGF1-CYC terminator was amplified, and then ligated with pPink-HC-scvSrh1(R)1-αpro-hIGF1 and pPink-HC-α_IGF1 treated with a restriction enzyme BamHI, thereby preparing a vector in which the promoter-gene-end signal was repeated two times. The whole process was repeated to obtain each of pPink-HC-6XSrh(R)1-αpro-hIGF1 and pPinkHC-6Xα-IGF1 cloned with a total of six promoter-gene-end signals.
Each of the two types of the vectors pPinkHC-6XSrh(R)1-αpro-hIGF1 and pPinkHC-6Xα-IGF1 prepared in Example 5 was linearized through cleavage with a restriction enzyme SpeI, a transformation was induced by introducing each vector into Komagataella phaffii by electroporation, the transformant was suspended in a YPDS (1% yeast extract, 2% Bacto peptone, 2% glucose, 1 M sorbitol) medium, and culture was performed for 2 hours while mixing the cells at 28° C. and 240 rpm.
Thereafter, the cells were plated on a minimal dextrose (MD) (1.34% yeast nitrogen base (YNB) without amino acids, 2% dextrose, and 1.5% Bacto agar) medium, the cells were stationary-cultured at 30° C. for 2 to 5 days, colonies whose diameters reached 1.5 to 2.0 mm first were selected, and the selected colonies were plated on the same medium again.
Subsequently, additional stationary culture was performed at 30° C. for 3 days, the grown single colony was obtained and inoculated into a buffered minimal glycerol (BMG) (100 mM potassium phosphate buffer, pH 5.4, 1.34% YNB, and 400 μg/L biotin, 2% glycerol) medium, and then, culture was performed at 30° C. for a day while mixing the colonies at 240 rpm.
Thereafter, the culture cells were inoculated into a buffered minimal methanol (BMM) (100 mM potassium phosphate buffer, pH 5.4, 1% yeast extract, 2% Bacto peptone, 1.34% YNB, and 400 μg/L biotin, 1% methanol) liquid medium so that an absorbance (OD600) was 0.5, and the cells were cultured in the same manner as that of Example 4.
After the culture for 3 days was completed, the human insulin-like growth factor-1 present in the medium was quantified by an ELISA kit. The results thereof are illustrated in
It was confirmed from this that the measured secretory expression levels according to the two signal sequences were similar to each other, and in the case where the hybrid signal sequence scvSrh1(R)1-αpro was used, the reproducibility was remarkably excellent because the difference in productivity for each batch was insignificant in comparison to the case where the signal sequence of the α-mating factor was used.
In order to confirm whether the difference in reproducibility as described above is also caused by a difference in the type of medium, the medium for secretory expression of hIGF1 was changed to a BMMY (100 mM potassium phosphate buffer, pH 5.4, 1% yeast extract, 2% Bacto peptone, 1.34% YNB, and 400 μg/L biotin, 1% methanol) liquid medium, and the secretion amount of hIGF1 was measured in the same manner as in 6.1 of Example 6.
As a result of measuring the secretion amount of hIGF1 in the medium by an ELISA kit after completion of the culture, it was confirmed that, similar to the BMM medium, in the case of the hybrid signal sequence scvSrh1(R)1-αpro, the reproducibility was significantly excellent and a concentration of 26.03 μg/mL of hIGF1 was exhibited, which showed that the production yield was about three times higher than that in the case where a concentration of hIGF1 was 8.68 μg/mL when the signal sequence of the α-mating factor as the control group was used (see
The pre-culture prepared in Example 6 was inoculated into a 3 L baffled flask containing 300 mL of a BMMY (100 mM potassium phosphate, pH 5.4, 1% yeast extract, 2% Bacto peptone, 1.34% YNB, and 400 μg/L biotin, 1% methanol) medium so that OD600 was 0.5, culture was performed at 30° C. for 3 days under mixing at 220 rpm, and 1% methanol was added every 24 hours.
After completion of the culture, the culture medium was centrifuged at 4° C. and 6,000 rpm for 10 minutes to remove cells.
The culture medium removed by filtration of remaining cells with a filter having a pore size of 0.45 um was diluted 3-fold to a pH 4.0 with a 20 mM sodium acetate buffer. Thereafter, the diluted culture medium was loaded on a 5 mL Hitrap SP cation exchange column (GE Healthcare) equilibrated with the same buffer using fast protein liquid chromatography (FPLC), and washing was sufficiently performed with a 20 mM sodium acetate buffer (pH 4.0) to a level at which UV absorption at 280 nm was not observed.
Thereafter, hIGF1 was eluted (50 mL in total) by inducing a concentration gradient from 0 to 100% using a 20 mM sodium acetate buffer to which 1 M NaCl was added.
The eluted fractions were subjected to 15% SDS-PAGE and western blot analysis using anti-hIGF1 mouse IgG antibodies (R&D System) and goat anti-mouse IgG (Elabscience (Houston, TX, USA)).
The results thereof are illustrated in
In the elution chromatogram, the peak 2 with a high absorbance was found adjacent to the fraction from which the hIGF1 was eluted, and it was presumed that a salt or pigment present in the medium was detected because there was no protein detected in SDS-PAGE.
It was confirmed from the results that the protein observed in SDS-PAGE through western blot was hIGF1. hIGF1 having a high purity of about 90% or more was purified by a single process using cation exchange chromatography, and 21 mg/L of hIGF1 (>80% recovery yield) was recovered from culture medium.
In the case of a recombinant protein for pharmaceutical use, modification of the protein primary sequence (amino acid sequence) acts as an important variable for re-clinical consideration. In the case of the signal sequence of the α-mating factor, after cleavage of the signal sequence during extracellular secretion, four amino acids (EAEA) remaining at the N-terminal region of the protein are generally removed by STE13 (peptidyl-dipeptidase A) in human cells. On the other hand, the presence or absence of this scar of the recombinant protein differs depending on the type of protein fused with the signal sequence of the α-mating factor.
In order to completely remove the four residual amino acids that were likely to remain from the recombinant protein recovered through the above Example, asparagine (N) or glycine/arginine (GR), which was an amino acid that was cleavable by hydroxylamine or thrombin, was inserted between the hybrid signal sequence and hIGF1 by the following method, respectively.
First, linear pPinkHC-scvSrh1(R)1_αpro-N-hIGF1 and pPinkHC-scvSrh1(R)1_αpro-GR-hIGF1 were amplified through a polymerase chain reaction using a set of primers, IGF GR/N 2F and IGF N 2R or IGF GR/N 2F and InIGF GR 2R, with pPinkHC-scvSrh1(R)1_αpro-hIGF1 as a template, and ligation independent cloning was performed, thereby obtaining recombinant plasmids (see
After confirming that there was no change except for the inserted base sequence by analyzing the base sequence of the cloned inserts, recombinant cells in which pPinkHC-6X scvSrh1(R)1_αpro-N-hIGF1 and pPinkHC-6X scvSrh1(R)1_αpro-GR-hIGF1 were transformed were obtained in the same manner as that of Example 5 (see
Thereafter, the productivity was measured by purifying hIGF1 by the methods of Examples 6 and 7 (see
From the results obtained by using the N and GR sequences additionally inserted into the amino terminus of hIGF1, it was confirmed that in the case where the hIGF1 was secreted and expressed using the hybrid signal sequence, the residual sequence was completely removed by introducing a new strategy for removing the residual sequence such as chemical cleavage or addition of a thrombin recognition sequence. In addition, although the exact mechanism was unknown, it was also confirmed that the hybrid signal sequence was completely removed during the analysis process using, as the control group, hIGF1 secreted by pPinkHC-6X scvSrh1(R)1_αpro-hIGF1 to which such a method was not applied.
As set forth above, according to an exemplary embodiment of the present invention, a heterologous signal sequence that significantly improves secretory expression of the human insulin-like growth factor-1 (hIGF1) is introduced into Komagataella phaffii, which is a unicellular eukaryotic cell, used as a host, such that various types of available signal sequences may be secured, and significantly improved secretory production may be achieved.
Special portions of contents of the present invention have been described in detail hereinabove, and it will be obvious to those skilled in the art that this detailed description is only an exemplary embodiment and the scope of the present invention is not limited by this detailed description. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0059486 | May 2022 | KR | national |
