CN118440975A - A method for preparing recombinant humanized collagen with triple helical structure expressed by yeast - Google Patents

Abstract

The invention discloses a preparation method of recombinant humanized collagen with a yeast expression triple helix structure, belonging to the technical field of genetic engineering. According to the invention, a plasmid capable of being used for in vitro multicopy and in vivo multicopy is constructed, a collagen expression vector is constructed based on the plasmid, a high expression strain is obtained through expression quantity screening, a P4H expression plasmid is introduced into the high expression strain to obtain a co-expression strain, and after fermentation and purification, the recombinant humanized collagen with a yeast expression triple helix structure can be obtained, wherein the recombinant humanized collagen contains hydroxyproline, and the hydroxyproline accounts for more than 30% of the total proline. The recombinant humanized collagen prepared by the invention can be widely applied to the fields of cosmetics, medical cosmetology, medical instruments, biomedical materials and the like.

Description

A method for preparing recombinant humanized collagen with triple helical structure expressed by yeast

Technical Field

The invention relates to the technical field of genetic engineering, and in particular to a method for preparing recombinant humanized collagen with triple helical structure expressed by yeast.

Background technique

Collagen is the main component of the extracellular matrix and the most abundant and widely distributed protein in animals. It has a unique tissue structure and biological function, and plays an important role in maintaining the normal physiological functions and injury repair of cells, tissues, and organs.

Recombinant humanized collagen is a recombinant protein that modifies or resynthesizes a gene sequence of human collagen and then expresses the new gene sequence through a host protein expression system. Recombinant human collagen has the advantages of collagen antigenicity, biodegradability, and good biocompatibility, and does not have the disadvantages of animal collagen, such as viral risks and immune rejection, so it is widely used in the field of biomaterials.

There are a large number of literature reports on cases of recombinant expression of humanized collagen and other collagens, such as "Recombinant Microbial Systems for the Production of Human Collagen and Gelatin" published by Julio Báez et al., and "Three Decades of Research on Recombinant Collagens" published by Fertala. Although both involve the construction of recombinant human collagen, they all involve recombinant human collagen single chains. Such recombinant collagen lacks key collagen characteristics, including triple helical conformation, modification of proline and lysine residues, and resistance to enzymatic degradation. Another example is "CN114853881B A recombinant human fusion collagen and its efficient hydroxylation method and application". In this patent, recombinant human collagen is co-expressed with the proline hydroxylase gene of Chlorella paramecium virus 1, but non-human P4H has different substrate specificity, and the obtained recombinant collagen may cause immune risks. That is, although a variety of recombinant collagens are disclosed in the prior art, they have the above-mentioned defects and cannot meet the diversified needs of the market, especially the market demand for triple helical recombinant humanized collagens. Therefore, the present invention intends to develop a yeast-expressed recombinant humanized collagen with a triple helical structure to achieve efficient expression of triple helical recombinant humanized collagens and meet the market demand for triple helical recombinant humanized collagens.

Summary of the invention

The purpose of the present invention is to provide a method for preparing a recombinant humanized collagen with a triple helix structure expressed by yeast to solve the problems existing in the above-mentioned prior art. A plasmid that can be used for multiple copies in vitro and multiple copies in vivo is constructed, and then a collagen expression vector is constructed based on the plasmid and a high-expression strain is obtained by expression amount screening. Finally, a P4H expression plasmid is introduced into the high-expression strain to obtain a co-expression strain, and a triple helix structure recombinant humanized collagen can be obtained after fermentation and purification.

To achieve the above object, the present invention provides the following solutions:

The present invention provides a method for preparing a recombinant humanized collagen with a triple helix structure expressed by yeast, comprising the following steps:

The recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III are transferred into yeast in any of the following ways (1) or (2):

(1) transferring any one of the recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III into yeast to obtain recombinant strain I; transferring any one of the recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III into the recombinant strain I to obtain recombinant strain II; transferring the P4H expression vector into the recombinant strain II to obtain recombinant strain III;

(2) Based on (1), any one of the recombinant expression plasmid I, the recombinant expression plasmid II and the recombinant expression plasmid III is transferred into the recombinant bacteria III to construct the recombinant bacteria IV;

Fermenting the recombinant bacteria III or the recombinant bacteria IV to obtain recombinant humanized collagen with a triple helical structure expressed by yeast;

Gene fragment I, gene fragment II or gene fragment III are used to transform pPIC9K vector respectively to construct plasmid I, plasmid II and plasmid III; the truncated sequence 5D-Cpro of the functional region of human type III collagen α1 chain is cloned into plasmid I, plasmid II and plasmid III respectively to obtain recombinant expression plasmid I, recombinant expression plasmid II and recombinant expression plasmid III; the nucleotide sequences of the gene fragment I, gene fragment II or gene fragment III are shown in SEQ ID NO:1-3 respectively.

Preferably, the construction method of the recombinant bacteria III comprises: transferring the recombinant expression plasmid I into yeast to obtain a recombinant strain I; transferring the recombinant expression plasmid II into the recombinant strain I to obtain a recombinant bacteria II; transferring the P4H expression vector into the recombinant bacteria II to obtain a recombinant bacteria III.

Preferably, the method for constructing the recombinant bacterium IV comprises: transferring the recombinant expression plasmid into the recombinant bacterium III to construct the recombinant bacterium IV.

Preferably, the P4H expression vector is obtained by replacing the KanR gene of the P4H 9K DP plasmid with the gene fragment shown in SEQ ID NO: 6.

Preferably, the nucleotide sequence of the human type III collagen α1 chain functional region truncated sequence 5D-Cpro is as shown in SEQ ID NO:5.

Preferably, the plasmid I is based on the pPIC9K vector as the backbone, and the 4648-9221 bp in the nucleotide sequence of the pPIC9K vector is replaced with the sequence shown in SEQ ID NO: 1, and the 938-943 bp in pPIC9K is deleted by point mutation, and the BamHI recognition site is introduced between 1583-1584 bp by point mutation.

Preferably, the plasmid II is obtained by replacing PpHIS4 on the plasmid I with the sequence shown in SEQ ID NO: 2;

The plasmid III is obtained by replacing PpHIS4 on the plasmid I with the sequence shown in SEQ ID NO: 3.

Preferably, the yeast includes Pichia pastoris KM71 and X33.

Preferably, the fermentation conditions are a rotation speed of 300 rpm, a ventilation volume of 4 L/min, a temperature of 30° C., and a pH of 6.0;

After the fermentation is completed, the fermentation liquid is centrifuged to collect the bacteria, the bacteria are resuspended at a mass-to-volume ratio of 1:10, crushed, centrifuged, and the precipitate is collected. The precipitate is washed, renatured, enzyme-digested, and freeze-dried to obtain recombinant humanized collagen with a triple helical structure expressed by yeast.

The present invention also provides a recombinant humanized collagen protein with a triple helix structure expressed by yeast, which is prepared by the preparation method.

The present invention discloses the following technical effects:

The present invention uses the modified plasmid to construct a recombinant humanized collagen expression vector, first screens the collagen expression to obtain a high-expression strain, then introduces the P4H expression plasmid to obtain a co-expression engineering strain, which can be used to study the hydroxylation differences of P4H from different sources on full-length collagen in Pichia pastoris. The fermentation and purification products of the co-expressed engineering strains are obtained by fermenting and purifying the mature yeast to express a recombinant humanized collagen with a triple helix structure, and the recombinant humanized collagen contains hydroxyproline, and the hydroxyproline accounts for more than 30% of the total proline.

The present invention provides a method for preparing recombinant humanized collagen with a triple-helix structure expressed by yeast. The modified plasmid can facilitate the construction of multi-copy collagen expression strains. The collagen obtained by fermenting the recombinant engineering strain has a triple-helix structure and can be used in scenarios requiring large molecular weight and high support in the fields of medical health and medical beauty.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a map of plasmid pPIC9K;

Fig. 2 is a map of plasmid pMChZ-AOX;

Fig. 3 is a map of plasmid pMCnZ-AOX;

Fig. 4 is a map of plasmid pMCrZ-AOX;

FIG5 is a map of the P4H expression vector pPIC9K-P4H(DP)-1;

Figure 6 shows the results of Western Blot experiments; A: WB detection results of collagen after 72h of induction with KM71H/5D-Cpro-1#-Z ^S ; B: Comparison of expression levels of KM71H/5D-Cpro-2# and KM71H/5D-Cpro-2#-P4H with KM71H/5D-Cpro-1#; C: Comparison of expression levels at different electroporation times;

In the embodiment of the present invention, the naming rule of the obtained transformants is: starting strain name/POI-number of electroporation #-screening antibiotic sensitivity, and the antibiotic sensitivity description will be omitted if the screening marker is not recovered. Here, Z is the abbreviation of Zeocin, S (sensitive) represents sensitive, R (resistant) represents resistance, the number before # represents the number of electroporation of the collagen expression plasmid, and the number with a circle represents the same plasmid electroporation as the previous round.

FIG7 shows the copy number determination results of different strains;

FIG8 is the circular dichroism spectrometer test result of 5D-Cpro lyophilized powder;

FIG9 is a transmission electron microscopy test of 5D-Cpro freeze-dried powder.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

The present invention constructs a plasmid that can be used for multiple copies in vitro and in vivo at the same time, then constructs a collagen expression vector based on the plasmid and obtains a high-expression strain by screening the expression amount, and finally introduces the P4H expression plasmid into the high-expression strain to obtain a co-expression strain; after fermentation and purification, a triple helical structure type III recombinant humanized small molecule collagen can be obtained. The following is further described with specific examples.

Example 1 A method for preparing a recombinant humanized collagen protein expressing a triple helical structure in yeast

1. Plasmid construction

The present invention transforms the plasmid to construct a plasmid pMChZ-AOX, a plasmid pMCrZ-AOX or a pMCnZ-AOX that can be used for multiple copies in vitro and multiple copies in vivo. The specific construction method is as follows:

1.1 Construction of plasmid pMChZ-AOX

The sequence of SEQ ID NO:1 was synthesized and replaced with bp 4648-9221 of the pPIC9K vector (purchased from Invitrogen, map see FIG1 ), i.e., the KanR resistance gene, Bom and AOX13’fragment elements in pPIC9K were replaced with the replacement fragment SEQ ID NO:1; BamHI (938-943 bp) and αMF located before αMF in pPIC9K were deleted by point mutation, and a BamHI recognition site was introduced downstream of AOX1tt (also called AOX1 transcription termination or AOX1 termination) (between 1583 and 1584 bp) by point mutation, while the sequences of other vector elements remained unchanged.

Among them, the synthesized SEQ ID NO:1 sequence contains lox71 and lox66 sequences at both ends, ori is located upstream of lox71, and a Cre transcription unit is located inside. The Cre gene is driven by the AOX1 promoter with a mutation in the SacⅠ restriction site; there are prokaryotic and eukaryotic promoters upstream of BleoR, which can be used for prokaryotic and eukaryotic screening, and there is a prokaryotic promoter upstream of AmpR, which can be used for prokaryotic screening.

The plasmid pMChZ-AOX (h represents the recombination site PpHIS4, and Z represents the screening marker BleoR) was constructed. The plasmid can express Cre recombinase when induced by methanol, catalyze the recombination of lox71 and lox66, and realize the recovery of resistance genes. BamHI located after AOX1tt can be used to construct in vitro multi-copy plasmids with the same tail enzyme. The original plasmid pPIC9K and the pMChZ-AOX plasmid constructed by the present invention are shown in Figure 2.

The meanings of the various parts in SEQ ID NO: 1 are explained as follows:

The lowercase letters (positions 61-649) are ori, the gray background at the 5' end (positions 782-815) is lox71, the gray background at the 3' end (positions 5384-5417) is lox66; the single underline "" (positions 854-1714) is AmpR, the double underline "" (1715-1819) is pAmpR (AmpR promoter), the wavy line (1854-2788) is the pAOX1 promoter with the mutated SacⅠ site, the dotted line "__" (2814-3842) is the Cre recombinase gene, the emphasis (3940-4186) is AOX1tt, and the bold underline (4200-4611th position) is pTEF1 (corresponding to TEF1 promoter in the figure), the wavy lowercase letter part (4619-4666th position) is pEM7 (corresponding to EM7 promoter in the figure), the italic lowercase letter part (4685-5059th position) is BleoR, and the underlined lowercase letter part (5125-5372th position) is CYC1tt (corresponding to CYC1 terminator in the figure). The sequence information of SEQ ID NO: 1 is as follows:

1.2 Construction of plasmid pMCnZ-AOX

The synthesized sequence SEQ ID NO: 2 (NTS) was substituted for PpHIS4 on the pMChZ-AOX obtained in 1.1 above to obtain the plasmid pMCnZ-AOX (n represents the recombination site is NTS), the map of which is shown in FIG3 .

The sequence information of SEQ ID NO:2 is:

ATCTAGAAGACCAGCTTCGTAAGCAATCTGGACAATTATGTAAGCGGGTTACGTAAACAGTTATGTAAGCAGAAAAATTTCAAACGACAAAACTTGGGGTCTACAGACACAGTAGCCAGAAGATTGCACTACCATTCGACTCCTCATGACCCACTCTTTCGATCCATGTAGTTAGGTTACCGTTTTTCCTAATATTTAAGGATGTTGAAAATTCATTTTCATTTTTTTCGTTTTTAAGATTTTCTCACAACTCTTCCAAAGATTACT AGTTGACTTTTCAAATATTTAGGGTATTTTTCTCACTTTTCCTAGCAAACTCCAATTGGTGGGTTTCAGTGCAATGGAGTATCACCTTGCAACCACAACGTAATAGCTAACTTGTGGCCACCATGTCTGGTTGTAGAGATAATTGGATTCTAATGTGGATCACATGACTACTCACGTGTCAAAAACCCAACCTGACTTGGCCCAGCTTAGCAAGAATATTTCGAATCCACTCTTGTGGCCTAGTGGACCGCTGCGTCTTCAA TGAC.

1.3 Construction of plasmid pMCrZ-AOX

The sequence of SEQ ID NO: 3 (PpRGI2) was synthesized to replace PpHIS4 on the pMChZ-AOX obtained in 1.1 above, and the plasmid pMCrZ-AOX (r represents the recombination site PpRGI2) was obtained, and the map is shown in FIG4 .

SEQ ID NO:3

GCTTCATACCCAGCATTGACCTTTGGTATGAGCATCTGAAAAACAACCAGGTGTTGCAAAGTTAAACATCCTTCTTTGTTCATATAGAACCCACTATTCATGGTACTCCCCAATCGAATTTCACATTCTGGTTTTGAAATTACACACCACGTTAGCTTATAAGATTTCATATAACTTATTGATATACGGTTTCCATTGTTCGAATAGTTGAGGTTGTATGTAATTCGATTGAAGGGGCCATTTTTGTTTCCTA CTTTTCCTGGGAGCTTATCCGATGCGCTTCAAAGCTGGAATTGTAAATATAGAGAAAAAGAAGGATGTTGTTTTATTCTTGAAAGAGTATAATTTTACTTCTAGCAACTCTCCCACTTCGCTTGACTTCATTTATTTCTTGGGCACATAGGCGTAGTAATCTAGACCAACAGATAATTTGCCGGAATGATATAGCGATTGGAAAATGAACTGAAATTTTTTGCTGTCTTTCAATTTGACGGGCAGTTCATCAGG CGCGCCGTGACCGACATATAAATACGTTGAGAATGTTATTCTTCCTCGTAGTTGAAGTGGCTTCATAATTTCAGAACTCAATAGATAAACTAGGATGTTTTAAAGCAATTAATGCTCACAAGTAAGGAGCGACTCTCTTGCTTTTCGAATACTAAAAGTATCGTCCCAACCCAGAAAAAAAGACCTCTTAACTGCAAAATAAACTCTATATATTTCTTCTAAAACAGTTTCAGGTTGGATAGTATCGCATTC TCATCACTTCTAACTAGTAGGCCATGAGATATATTAACGTTTACTTGAGTCTCTAAGTTCTCCGAATTAGATGCACAGCACAAACAAGATTAGGTTTCACTTGGTACAAAATACGAACAGAGTTTAAGGTCGTAATTTCATTTCGTTATTGATCCCCACAATCTATTCTTATCACAGTCATCAGATAGTCGCGAAAAAGCATGCAGAAAAGGGGGTCGTCCCTATCTAAGTTGTAGCATTACAACAATGACC.

2. Obtaining recombinant humanized collagen and its expression vector

The full-length sequence of type III collagen α1 chain was optimized to obtain the truncated sequence 5D-Cpro of the α1 chain functional region, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleic acid sequence SEQ ID NO: 5 was obtained by reverse translation according to the Pichia codon table, and the GC content and secondary structure were optimized. 5D-Cpro was cloned into pMChZ-AOX and pMCrZ-AOX, respectively, to obtain the recombinant expression plasmids pMChZ-5D-Cpro and pMCrZ-5D-Cpro. 5D-Cpro was cloned into pMCnZ-AOX to obtain the recombinant expression plasmid pMCnZ-5D-Cpro.

SEQ ID NO:4 sequence information is:

LEKRAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSP GPAGQQGAIGSPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHP GPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPCCGENLYFQGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDS SAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVQLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFLHHHHHH.

SEQ ID NO:5 sequence information is:

TTAGAGAAGCGAGCAGGAAATACCGGGGCACCGGGCTCACCTGGCGTGAGCGGTCCCAAGGGCGACGCCGGACAGCCGGGTGAAAAGGGCTCCCCCGGTGCTCAAGGGCCTCCTGGGGCACCGGGTCCGTTGGGAATAGCTGGGATAACCGGAGCGAGGGGCTTGGCAGGACCCCCAGGAATGCCGGGCCCAAGGGGCTCTCCTGGTCCTCAGGGAGTAAAAGGGGAAAGTGGGAAACCTGGTGCAAATGGTCTTTC CGGGGAGAGAGGACCCCCGGGGC CTCAAGGGGCTTCCTGGCCTAGCCGGTACAGCGGGTGAACCGGGCCGCGATGGCAACCCCGGCAGTGACGGTTTACCTGGACGGGATGGTTCTCCCGGCGGCAAGGGGGATAGAGGGGAAAACGGGTCGCCGGGGGCGCCTGGAGCGCCTGGGCATCCCGGTCCCCCCGGGCCCGTTGGCCCGGCGGGCAAGAGCGGTGACCGAGGTGAGTCGGGGCCAGCCGGTCCTGCTGGTGCTCCTGGACCTGCTGGCTCTCG TGGTGCTCCTGGACCGCAGGGACC AAGAGGTGATAAGGGGGAGACTGGGGAGCGGGGGGCAGCGGGAATCAAAGGACACCGAGGTTTCCCCGGGAACCCTGGGGCTCCAGGTTCGCCTGGTCCTGCTGGACAGCAAGGAGCCATTGGTTCGCCAGGCCCCGCTGGGAATACCGGAGCCCCGGGTAGCCCGGGTGTGTCAGGCCCCAAGGGAGATGCGGGCCAACCGGGCGAAAAAGGTTCTCCAGGGGCTCAGGGACCCCCCGGAGCCCCGGGACCGTTAGGCATA GCGGGGATAACGGGAGCA AGAGGCCTGGCAGGACCACCAGGAATGCCGGGCCCGCGAGGTAGCCCAGGTCCCCAAGGAGTAAAAGGTGAGAGCGGTAAACCAGGAGCAAATGGCTTGTCAGGCGAACGGGGGCCGCCAGGGCCACAAGGCCTACCTGGTCTTGCTGGGACTGCAGGGGAACCAGGGCGCGACGGTAATCCCGGCAGCGACGGACTACCTGGACGTGATGGCTCCCCCGGAGGCAAGGGGGACCGCGGGGAGAACGGTAGTCCTG GTGCCCCAGGAGCACCTGGTCATCC AGGACCGCCGGGTCCTGTGGGTCCCGCCGGCAAGTCAGGGGATAGGGGCGAAAGTGGGCCGGCCGGACCCGCAGGGGCTCCAGGACCCGCCGGCTCCCGTGGCGCGCCAGGACCGCAAGGACCACGGGGAGATAAGGGTGAGACAGGAGAGCGGGGGGCTGCAGGCATAAAAGGTCACCGTGGTTTCCCGGGTAACCCGGGTGCCCCGGGATCACCAGGGCCTGCTGGGCAGCAAGGAGCAATTGGATCACCAGGGACCAGCT GGTCCTTGCTGCGGAGAG AACCTGTACTTCCAGGGAGGGGTGGGCGCAGCGGCGATTGCGGGTATAGGGGGCGAGAAGGCGGGTGGATTTGCTCCCTATTATGGCGACGAACCAATGGACTTCAAAATTAACACTGATGAAATAATGACCTCTTTTAAAGTCGGTCAATGGACAAATTGAGTCTCTAATTTCCCCCGACGGCTCGCGTAAGAACCCTGCACGCAACTGCCGTGATTTAAAATTTTGCCACCCCGAGTTGAAATCCGGCGAATACTGGGTTGA CCCTAATCAAGGGTGTA AACTTGACGCGATCAAAGTTTTCTGTAATATGGAAACAGGAGAGACGTGTATCTCCGCGAACCCTCTTAACGTCCCGCGGAAACACTGGTGGACAGACTCCAGTGCCGAAAAAGCACGTATGGTTCGGGGAATCTATGGATGGTGGCTTTCAATTCTCATACGGTAATCCCGAACTGCCTGAAGATGTTCTCGATGTCCAGCTCGCCTTTCTTAGGCTCCTGTCGAGTCGAGCCAGTCAAAACATCACTTATCACT GTAAAAATTCTATTGCTTACAT GGACCAGGCGAGCGGGAACGTTAAAAAGGCCTTGAAGTTAATGGGTAGTAATGAGGGGGAGTTTAAAGCAGAAGGTAATTCTAAATTTACTTATACGGTCCTCGAGGACGCTGTACGAAACATACGGGCGAATGGAGCAAGACCGTGTTTGAATACAGGACAAGAAAGGCGGTCGTATCGCACCCTATGATATCGGTGGGCCAGATCAGGAATTTGGGGTAGATGTAGGGCCAGTCTGC TTCCTGCATCATCATCACCACCAT.

Construction of P4H expression vector pPIC9K-P4H(DP)-1:

Synthesize SEQ ID NO: 6 to replace KanR of P4H 9K DP plasmid in CN114480471A, i.e. add P _TEF and P _EM7 to the 5' end of the selection marker (according to the direction of pPIC9K plasmid), and add CYC1tt to the 3' end to obtain P4H expression vector pPIC9K-P4H (DP) -1, the plasmid spectrum of which is shown in Figure 5.

The sequence information of SEQ ID NO: 6 is as follows, wherein the bold underlined uppercase letter part (positions 1203-1614) is _PTEF , the shaded bottom (positions 1148-1195) is _PEM7 , the thin underlined lowercase letter part (positions 314-1129) is KanR, and the ununderlined lowercase letter part (positions 1-248) is CYC1tt):

3. Construction of recombinant engineering strains, induced expression and strain screening

(1) Construction of engineered strains

Pichia KM71 was used as the starting strain. Here, KM71 was used as an example. The competent state of Pichia was prepared according to the competent state preparation method described by Lin-Cereghino (Lin-Cereghino et al, 2005). The pMChZ-5D-Cpro obtained above was linearized using SalⅠ to obtain the pMChZ-5D-Cpro linearized plasmid. The pMChZ-5D-Cpro linearized plasmid was electroporated into the competent state of Pichia according to the method of Invitrogen's Pichia Expression Kit USER GUIDE, and then coated on a YPDZ (bleomycin, 300 μg/mL) plate to obtain the strain KM71H/5D-Cpro-1#-Z ^R . The screening marker was recovered to obtain the strain KM71H/5D-Cpro-1#-Z ^S . (Wherein, Z ^R indicates bleomycin resistance, Z ^S indicates bleomycin sensitivity; the number before # indicates the number of electroporation of the collagen expression plasmid).

pMCnZ-5D-Cpro was linearized with SpeⅠ and then electroporated into KM71H/5D-Cpro-1#-Z ^S to obtain KM71H/5D-Cpro-2#-Z ^R . The selection marker was recovered to obtain strain KM71H/5D-Cpro-2#-Z ^S .

pPIC9K-P4H(DP)-1 was linearized with SalⅠ and then electroporated into KM71H/5D-Cpro-2#-Z ^S to obtain KM71H/5D-Cpro-2#-Z ^S -P4H.

pMCrZ-5D-Cpro was linearized with SpeⅠ and then electroporated into KM71H/5D-Cpro-2#-Z ^S -P4H to obtain KM71H/5D-Cpro-3#-P4H.

(2) Inducible expression

The engineered strain obtained above was inoculated into a 100mL Erlenmeyer flask containing 10mL of BMGY medium, and cultured at 28-30°C and 220rpm until OD ₆₀₀ was 2-6 (16-18h). Centrifuge at 1500-3000g for 5min at room temperature, collect the bacteria, resuspend the bacteria with BMMY medium to about OD ₆₀₀ of 2, place on a shaker at 28-30°C and 220rpm to continue growing for 3 days, and add 100% methanol to the culture medium every 24h to a final concentration of 1.0%. After methanol induction, collect bacterial liquid samples every 24h, the sample volume is 1mL, placed in a 1.5mL EP tube, centrifuged at 12000g for 5min at 4°C, and the supernatant and bacteria are collected separately. Take 80μL of the supernatant, add 20μL of loading buffer, heat at 80°C for 5min to prepare the sample, and take 20μL for SDS-PAGE. Add 200 μL of cell wall breaking solution and 40% of the liquid volume of 0.5 mm glass beads to the bacteria; vortex for 1 min, place on ice for 1 min, and cycle 6-10 times; centrifuge at 12000g for 30 min at 4°C, take 80 μL of the supernatant, add 20 μL of loading buffer, heat at 99°C for 5 min to prepare the sample, dissolve the precipitate with 8M urea, prepare the sample according to the supernatant operation steps, take 20 μL for SDS-PAGE, and then perform Western Blot. The detection results are shown in Figure 6. KM71H/5D-Cpro-1#-Z ^R was induced for 72 hours and the expression was identified with anti-6His antibody as the primary antibody. There was a target band in the cell, and the results are shown in Figure 6 A. The strain that correctly expressed the target protein was used as the starting strain. The 5D-Cpro expression plasmid with the recombination site was replaced by electroporation twice and the P4H expression plasmid was co-transfected. The target protein was identified by anti-6His antibody and P4HA1 antibody. The expression level of 5D-Cpro in the strain co-transfected with P4H was significantly improved. The four P4H co-expression strains screened out could correctly express P4HA and P4HB. The results are shown in Figure 6B. As shown in Figure 6C, the expression level of different electroporation times is compared. KM71H/5D-Cpro-2#-Z ^S -P4H has the highest expression level, exceeding KM71H/5D-Cpro-3#-P4H.

4. Identification of target gene copy number

The strains related to 5D-Cpro were entrusted to Shanghai Sangon Biotech to detect the copy number of the target gene using digital PCR, and the PCR instrument was BIO-RAD's T100. The copy number of the target gene in the strain obtained by the first electroporation of the target gene was 1, and the ratio of the copy number of the target gene in other strains to that of the strain was plotted, as shown in Figure 7. The copy number of the target gene in the 5D-Cpro related strains was consistent with the expression level detected by WB. The copy number of KM71H/5D-Cpro-2#-P4H was about 2.5 times that of the control. After 3 rounds of electroporation, the expression level decreased, and the corresponding copy number was also low. Although the plasmid recombination site was replaced, the target gene was still lost.

5. High-density fermentation and protein purification

The engineered bacteria with high protein expression levels identified were cultured in a fermenter for high-density fermentation and protein purification.

KM71H/5D-Cpro-2#-P4H was inoculated into a 1L shake flask containing 200mL of seed culture medium YPG, and cultured at 220rpm and 30°C for 18-20h until _OD600 =2-10.

Use a 5L fermentation tank (Baoxing Biotechnology) with 2L fermentation medium. Before inoculation, adjust the speed to 300rpm, the ventilation volume to 4L/min, the temperature to 30℃, and adjust the pH with alkali solution prepared with concentrated ammonia water to set the pH to 6.0. First, add 0.9mL PTM1 Pichia yeast trace element mother solution, then add the prepared 200mL seed solution into the tank (flame circle inoculation), then click the dissolved oxygen electrode to calibrate, and start fermentation after calibration. When the dissolved oxygen drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain 30%; wait for the glycerol to be consumed, the dissolved oxygen rebounds, and the dissolved oxygen is greater than 70% (OD ₆₀₀ value is about 20), cancel the dissolved oxygen cascade speed, increase the stirring speed to 650rpm, and use 30% linkage feeding for glycerol, and feed 150mL. Stop feeding glycerol, and after the dissolved oxygen rebounds to more than 70%, induce culture with methanol, and the feeding rate is 4mL/h constant feeding. After 80 to 90 hours of induction, if the _OD600 value does not change significantly or decreases, the tank can be released to obtain the fermentation liquid.

After the fermentation, the bacterial liquid was centrifuged at 5000rpm for 30min to collect the bacterial cells, which were resuspended in the purified cell wall breaking solution at a ratio of 1:10 (W:V), broken by a homogenizer at 1000bar, and centrifuged at 12000rpm and 4℃ for 30min to separate the supernatant and precipitate. The precipitate was washed, renatured, digested with pepsin, ultrafiltered and desalted, and then freeze-dried to obtain 5D-Cpro freeze-dried powder.

6. Structural characterization of 5D-Cpro lyophilized powder

The 5D-Cpro lyophilized powder was reconstituted and then acid hydrolyzed. The sample was analyzed by liquid chromatography-mass spectrometry. The peak area of the targeted data was calculated using MassLynx quantitative software. The identification result was obtained using the standard curve method. The results showed that the hydroxyproline content reached more than 30% of the total proline.

The 5D-Cpro freeze-dried powder was dissolved in water and tested by circular dichroism spectrometer. The test results are shown in Figure 8, which are consistent with the circular dichroism spectral characteristics of triple-helical collagen.

The collagen sample was negatively stained and then tested by transmission electron microscopy. The test results are shown in FIG9 , which show the typical structural characteristics of collagen microfibrils with alternating light and dark features.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (10)

1. The preparation method of the yeast-expressed triple-helix recombinant humanized collagen is characterized by comprising the following steps of:

transferring the recombinant expression plasmid I, the recombinant expression plasmid II and the recombinant expression plasmid III into saccharomycetes in any one of the following modes (1) or (2):

(1) Transferring any one of the recombinant expression plasmid I, the recombinant expression plasmid II and the recombinant expression plasmid III into saccharomycetes to obtain a recombinant strain I; transferring any one of the recombinant expression plasmid I, the recombinant expression plasmid II and the recombinant expression plasmid III into the recombinant strain I to obtain recombinant bacteria II; transferring the P4H expression vector into the recombinant bacterium II to obtain recombinant bacterium III;

(2) Based on the step (1), transferring any one of the recombinant expression plasmid I, the recombinant expression plasmid II and the recombinant expression plasmid III into the recombinant bacterium III to construct a recombinant bacterium IV;

Fermenting the recombinant bacterium III or the recombinant bacterium IV to obtain recombinant humanized collagen with a yeast expression triple helix structure;

Respectively modifying pPIC9K vectors by using gene fragment I, gene fragment II or gene fragment III to construct plasmid I, plasmid II and plasmid III; cloning the truncated sequence 5D-Cpro of the human III type collagen alpha 1 chain functional region to a plasmid I, a plasmid II and a plasmid III respectively to obtain a recombinant expression plasmid I, a recombinant expression plasmid II and a recombinant expression plasmid III; the nucleotide sequences of the gene fragment I, the gene fragment II or the gene fragment III are respectively shown in SEQ ID NO. 1-3.

2. The method of claim 1, wherein the method of constructing recombinant bacteria III comprises: transferring the recombinant expression plasmid I into saccharomycetes to obtain a recombinant strain I; transferring the recombinant expression plasmid II into the recombinant strain I to obtain recombinant bacteria II; and transferring the P4H expression vector into the recombinant bacterium II to obtain recombinant bacterium III.

3. The method of claim 1, wherein the method of constructing recombinant bacteria IV comprises: and transferring the recombinant expression plasmid into the recombinant bacterium III to construct a recombinant bacterium IV.

4. The method of claim 1, wherein the P4H expression vector is obtained by replacing the KanR gene of the P4H 9K DP plasmid with the gene fragment shown in SEQ ID NO. 6.

5. The preparation method of claim 1, wherein the nucleotide sequence of the truncated sequence 5D-Cpro of the alpha 1 chain functional region of the human type III collagen is shown in SEQ ID NO. 5.

6. The method of claim 1, wherein plasmid I is obtained by using pPIC9K vector as skeleton, replacing 4648-9221bp in nucleotide sequence of the pPIC9K vector with SEQ ID NO. 1, deleting 938-943bp in pPIC9K by point mutation, and introducing BamHI recognition site between 1583-1584bp by point mutation.

7. The method of claim 1, wherein plasmid II is obtained by replacing PpHIS4 on plasmid I with the sequence shown in SEQ ID NO. 2;

The plasmid III is obtained by replacing PpHIS4 on the plasmid I with a sequence shown in SEQ ID NO. 3.

8. The method of claim 1, wherein the yeast comprises pichia pastoris KM71 and X33.

9. The process according to claim 1, wherein the fermentation is carried out at a speed of 300rpm, a ventilation of 4L/min, a temperature of 30℃and a pH of 6.0;

After fermentation, centrifugally collecting thalli from fermentation liquid, re-suspending thalli according to the mass-volume ratio of 1:10, crushing, centrifuging, collecting precipitate, washing, renaturating, enzyme cutting and freeze-drying the precipitate to obtain the recombinant humanized collagen with the yeast expression triple helix structure.

10. A recombinant humanized collagen having a yeast-expressed triple helix structure, characterized in that it is produced by the production method according to any one of claims 1 to 9.