CN111087478B - Recombinant collagen and preparation method thereof - Google Patents

Recombinant collagen and preparation method thereof Download PDF

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CN111087478B
CN111087478B CN202010028092.XA CN202010028092A CN111087478B CN 111087478 B CN111087478 B CN 111087478B CN 202010028092 A CN202010028092 A CN 202010028092A CN 111087478 B CN111087478 B CN 111087478B
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CN111087478A (en
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黄岩山
陈永露
方井晋
董佳里
杨程程
方艺琳
邹建文
章宏塔
徐德雨
黎常魁
金黎达
符威灵
何荣强
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Hangzhou Jiayan Biomedical Technology Co.,Ltd.
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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Abstract

The invention discloses a recombinant collagen and a preparation method thereof, and the amino acid sequence is shown as SEQ ID NO. 1. The recombinant collagen can be expressed and prepared in high-grade eukaryotic cells such as yeast and the like, has the property close to natural collagen, and can effectively support cell adhesion.

Description

Recombinant collagen and preparation method thereof
Technical Field
The invention relates to the field of protein, and more particularly relates to recombinant collagen and a preparation method thereof.
Background
Collagen is one of the most important and abundant proteins in mammals. The structure is characterized by (Gly-Xaa-Yaa) n, wherein Xaa and Yaa are proline frequently. Collagen generally has a triple helical structure and is insoluble in water, thus providing an insoluble scaffold for maintaining the shape and morphology of an organism; it is also responsible for the attachment of macromolecules, glycoproteins, hydrated polymers and inorganic ions, and even cell attachment. In addition, this is due to extensive post-translational modifications of collagen both before and after deposition on the extracellular matrix, for example Yaa is more commonly found in the hydroxyproline form. The ratio of the proline to hydroxyproline residues affects the melting point of collagen. The difference between the amino acid compositions at Xaa and Yaa positions also influences the characteristics of hydrophilicity, isoelectric point, secondary structure and the like of collagen.
Traditionally, collagen has been prepared by hot acid or alkali extraction of animal bones and skins, and this processed product is called gelatin. Thus, gelatin is essentially denatured and partially degraded collagen. Natural gelatin has a wide variety of uses, and in addition to being used primarily as a gelling agent in food products, gelatin is also used in medical and industrial applications, such as intravenous infusion, matrix implants, and the like. However, collagen hydrolysates of animal origin present a serious risk of viral infections, such as Bovine Spongiform Encephalopathy (BSE). Furthermore, the administration of gelatin of animal origin to humans also presents biocompatibility problems. Therefore, collagen or gelatin, which can be precisely controlled and reproduced in chemical composition and molecular weight, will be a safer and more efficient choice in a particular application field.
Disclosure of Invention
The invention aims to provide a preferable recombinant collagen and a preparation method thereof. The sequence of the recombinant collagen is shown in SEQ ID NO. 1.
As is well known, the collagen structural unit is Gly-Xaa-Yaa, and the highly repetitive and highly modified sequence is extremely difficult to prepare in Escherichia coli, so that the collagen or gelatin is generally expressed in yeast (such as Pichia pastoris) in recombination. However, natural and engineered yeast strains lack the complete post-translational modification system of mammalian cells, and therefore the expressed collagen or gelatin is modified to a much lesser extent than proteins of natural origin, especially hydroxyproline modifications at the Yaa position. Non-hydroxylated (non-hydroxylated) modified collagens have no gelling properties (MARC W.T. WERTEN et al, Yeast, 15, 1087-. Thus, the so-called collagen produced by expression in this manner does not have properties close to those of native collagen. In addition, if a hydroxylase (prolyl-4-hydroxylase) is simultaneously expressed in Yeast cells, it results in the failure of collagen secretion into the cell matrix due to the formation of a triple helix structure (MARC W.T.WERTEN et al, Yeast, 15, 1087-.
The invention provides a preferable recombinant gelatin protein, which can secrete collagen with gelation property when being expressed in yeast without hydroxylating enzyme, has extremely high expression quantity, and simultaneously has excellent stability and enzyme-resistant stability; in one embodiment of the present invention, the recombinant collagen has excellent properties of promoting mammalian cell adhesion.
The amino acid sequence of SEQ ID NO.1 is as follows:
Figure GDA0003467410370000021
application of recombinant collagen
The Gly-Xaa-Yaa unit of the recombinant collagen sequence provided by the invention is selected from units with high occurrence frequency in natural collagen, has the property close to that of the natural collagen sequence and high recombinant expression efficiency, and can be applied to the aspects of cosmetics, skin care products, food additives, industrial industries and the like.
Drawings
FIG. 1 comparison of the adhesion effects of recombinant gelatin protein on mammalian cells
Detailed Description
The invention will be further elucidated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are fully described in the following documents: for example, Molecular cloning, A Laboratory Manual, third edition (Sambrook J, Russell DW, Molecular cloning: A Laboratory Manual.3rd edition, New York: Cold Spring harbor Laboratory Press, 2001); protein purification: principles and practices 3rd edition (Scopes RK, Protein Purification: Principles and Practice,3rd edition, New York: Springer-Verlag, 1994), or, alternatively, may be performed according to instructions provided by the manufacturer of the reagents. The operation of the Pichia pastoris was carried out in accordance with the Invitrogen Pichia Expression Kit and Pichia Fermentation Process Guidelines, unless otherwise specified. In all the sequences below, the underlined parts are enzyme cleavage recognition sites, and the italicized parts are signal peptide sequences, unless otherwise specified.
Example 1: expression and purification of recombinant gelatin protein
The gene of the recombinant gelatin protein (SEQ ID NO:1) of the invention is chemically synthesized. The α factor signal peptide sequence (with XhoI site) and EcoRI recognition site of yeast GS115 were added at the 5' end during synthesis and cloned into the expression vector pPIC9(life technology), the ligation product transformed e.coli DH5 α competent cells and positive clones identified. The methanol yeast Pichia pastoris GS115 (His)-) For expression of the host bacteria, the obtained pPIC9 positive clone plasmid was linearized by electrotransformation and transformed into GS 115. After 3 days at 30 ℃ until single colonies appeared.
The recombinant yeast single colony transformed above is inoculated into 10ml BMGY liquid medium, 30 ℃, 250rpm culture 24 hours, then left overnight, the supernatant is discarded, 10ml BMMY liquid medium containing 1% methanol is added, 30 ℃, 250rpm induction expression. Selecting a strain with relatively high expression as an expression strain.
The expression strain obtained by screening was inoculated into a liquid YPD medium, and cultured overnight at 30 ℃ and 250rpm with shaking to OD600About 20, and the obtained product is used as seed liquid in the tank. The cultured seed solution was inoculated into a B.BRAUN BIOSTAT C-10 fermenter, and the medium was prepared according to the Pichia Fermentation Process Guidelines of Invitrogen. The inoculation amount is 10%, the fermentation temperature is set to be 30 ℃, the pH value is 5.0, and methanol is fed for induction expression when the glycerol is exhausted. The fermentation temperature is controlled at 25 ℃ in the expression stage, and the fermentation is induced for 72 hours and put in a tank.
High speed centrifugation is carried out to remove thalli, 1 liter of fermentation supernatant is taken, ice-precooled acetone is added at the temperature of 4 ℃ until the final concentration is 40-60 percent, the mixture is stirred for 30 minutes, centrifugation is carried out, and the precipitate is discarded. Adding ice-precooled acetone into the supernatant till the final concentration is 80%, stirring for 30 minutes, centrifuging and collecting the precipitate. The obtained pellet of recombinant gelatin-like fusion protein was resuspended in 100 ml of purified water and dialyzed against 20mM PB, pH7.0, at 4 ℃ overnight.
The finished recombinant gelatin solution was dialyzed, loaded onto a Q Sepharose FF column (GE Healthcare, XK26/20, column volume 50ml) equilibrated in advance with buffer A (20mM PB, pH7.0), and after completion of loading, unbound protein was eluted with 2 column volumes of buffer A followed by a linear gradient of 10 column volumes, 0-100% buffer B (20mM PB, 0.5M NaCl, pH 7.0).
The eluted recombinant gelatin protein was concentrated by ultrafiltration (Millipore, MWCO 10KD) to a protein concentration of about 10mg/ml, then desalted using a Sephadex G25 column (GE Healthcare, XK 26/20; column volume 50ml) with a buffer of 10mM PB, pH7.0, and freeze-dried. Protein concentration was determined by A280-standard concentration and purity was checked by 10% SDS-PAGE. The recombinant gelatin protein is negatively charged and highly hydrophilic, so that the recombinant gelatin protein is low in SDS combination efficiency, and is in a dispersed state on SDS-PAGE, and the apparent molecular weight is far greater than the theoretical molecular weight.
Example 2 gelation assay of recombinant collagen
Viscosity and reversible gelation in aqueous solution with respect to temperature are the most important properties of natural gelatin. When an aqueous solution of natural gelatin having a concentration of more than 0.5% is cooled to about 35-40 ℃, it first increases in viscosity and then forms a gel. The rigidity or strength of the gel depends on the GELATIN concentration, the inherent strength of the GELATIN, the pH, the temperature and the presence of additives (GELATIN hand book, GMIA, 2012). In this example, the congealing strength and the Boehringer's viscosity of the sample are determined by referring to the method of national standard food additive gelatin GB 6783-94. The freezing strength is measured by a freezing force tester, and the viscosity is measured by an ND-2 Bosch viscometer.
Recombinant collagen (10mg/ml), commercial collagen (10mg/ml) and BSA protein (10mg/ml) were prepared and left overnight (5 replicates) at 37 deg.C, 25 deg.C and 4 deg.C. The data in table 1 show that the recombinant collagen expressed by the present invention has gelation properties similar to those of commercially available collagen.
TABLE 1 Freeze Strength and viscosity of recombinant gelatin proteins
Sample (I) Freezing Strength (Bloom g) Viscosity (mPa.s)
Recombinant collagen (37 ℃ C.) 182 17
Recombinant collagen (25 ℃ C.) 197 18
Recombinant collagen (4 ℃ C.) 179 17
Commercially available collagen 189 16
BSA <5 <1
Example 3 cell adhesion assay
3mg/mL of recombinant collagen (dissolved in PBS buffer, pH 7.4) was added to a 96-well microplate at 300. mu.L per well, while Bovine Serum Albumin (BSA) solution, commercially available collagen (C520780, Biotechnology (Shanghai) Co., Ltd.) and PBS buffer were used as control groups, 3 wells per group. Incubate at 4 ℃ for 12h to allow protein adsorption to the well surface. PBST buffer wash to remove excess protein. CHO cells (DMEM/F-12 medium containing 10% FBS) were seeded at a density of 20,000 cells/well in coated 96-well plates described above at 5% CO2After culturing at 37 ℃ for 48 hours, the cell confluence rate was calculated (the confluence degree of commercially available collagen was 100%, FIG. 1).
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Zhejiang Dalong Biotechnology Ltd
<120> a recombinant collagen and a method for preparing the same
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 318
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
1 5 10 15
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Gly Glu Ser Gly Pro
20 25 30
Glu Gly Ser Glu Gly Glu Arg Gly Thr Ser Gly Pro Ser Gly Pro Glu
35 40 45
Gly Gln Asn Gly Glu Arg Gly Ser Pro Gly Pro Ser Gly Pro Glu Gly
50 55 60
Pro Ala Gly Ser Glu Gly Ala Pro Gly Pro Glu Gly Glu Ser Gly Pro
65 70 75 80
Ala Gly Glu Arg Gly Thr Ser Gly Pro Ser Gly Pro Glu Gly Gln Asn
85 90 95
Gly Glu Ser Gly Pro Asn Gly Pro Lys Gly Pro Glu Gly Glu Ser Gly
100 105 110
Pro Ala Gly Pro Pro Gly Ser Pro Gly Glu Arg Gly Pro Glu Gly Glu
115 120 125
Ser Gly Pro Ala Gly Pro Glu Gly Ala Glu Gly Pro Glu Gly Ser Pro
130 135 140
Gly Glu Pro Gly Pro Pro Gly Pro Ser Gly Pro Pro Gly Glu Pro Gly
145 150 155 160
Pro Ser Gly Ser Pro Gly Pro Pro Gly Pro Glu Gly Ser Pro Gly Pro
165 170 175
Pro Gly Pro Ser Gly Pro Ser Gly Ser Pro Gly Pro Glu Gly Glu Ser
180 185 190
Gly Pro Ala Gly Glu Arg Gly Thr Ser Gly Pro Ser Gly Pro Glu Gly
195 200 205
Gln Asn Gly Ser Glu Gly Glu Arg Gly Glu Glu Gly Pro Ser Gly Pro
210 215 220
Glu Gly Gln Asn Gly Glu Arg Gly Ser Pro Gly Glu Arg Gly Thr Ser
225 230 235 240
Gly Pro Ser Gly Pro Glu Gly Gln Asn Gly Pro Ser Gly Ser Glu Gly
245 250 255
Glu Ser Gly Pro Gln Gly Glu Lys Gly Ser Pro Gly Pro Glu Gly Glu
260 265 270
Ser Gly Pro Ala Gly Ser Glu Gly Pro Ser Gly Pro Ala Gly Pro Arg
275 280 285
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
290 295 300
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
305 310 315

Claims (1)

1. A recombinant collagen, wherein the amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 1.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110029111A (en) * 2019-01-30 2019-07-19 江苏悦智生物医药有限公司 Pichia pastoris produces the single-stranded method of recombination human source typeⅡ Collagen

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EP1870460B9 (en) * 2005-03-31 2012-12-05 Fuso Pharmaceutical Industries, Ltd. Methods of producing proteins having triple-helix structure
EP2454367B1 (en) * 2009-07-17 2013-10-16 The Texas A&M University System Designer collagens and uses thereof

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Publication number Priority date Publication date Assignee Title
CN110029111A (en) * 2019-01-30 2019-07-19 江苏悦智生物医药有限公司 Pichia pastoris produces the single-stranded method of recombination human source typeⅡ Collagen

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chain A,collagen triple helix-Protein;NCBI;《NCBI》;20020213;全文 *
Crystal structure of a collagen-like polypeptide with repeating sequence Pro-Hyp-Gly at 1.4 A resolution: implications for collagen hydration;Berisio R;《Biopolymers》;20010925;第56卷(第1期);第9页右栏第4段 *

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