CN118185979A - Method for improving hydroxylation rate of recombinant human collagen - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for improving hydroxylation rate of recombinant human collagen, which comprises the following steps: selecting a full-length collagen nucleic acid sequence, connecting the full-length collagen nucleic acid sequence to an expression vector containing an AOX promoter, constructing a THI11 double-promoter expression vector, and connecting a nucleic acid sequence capable of encoding human hydroxylase to form a human hydroxylase expression vector; the two expression vectors are jointly transformed into pichia pastoris to be screened to obtain recombinant expression strains capable of co-expressing the human collagen and the hydroxylase; performing induction culture on recombinant expression strains, and regulating two inducers to obtain recombinant human collagen with high hydroxylation rate; compared with the prior art, the invention realizes the independent regulation and control of the human collagen and the hydroxylase, improves the proportion of the hydroxy preserved amino acid in the recombinant human collagen, and obtains the recombinant human collagen with good stability.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for improving hydroxylation rate of recombinant human collagen.
Background
Collagen is an important protein in animals, is widely distributed in tissues such as skin, cartilage and blood vessels, participates in migration, differentiation and reproduction of cells, plays an important role in maintaining normal physiological functions of the cells, the tissues and organs, and is widely applied in the fields of food, feed, beauty, cosmetics, medicines and the like.
Collagen is composed of peptide triplets Gly-X-Y, where X and Y can theoretically be any amino acid. However, proline has a preference for the X position, while hydroxyproline has a preference for the Y position. Hydroxyproline is formed by hydroxylating proline by the action of proline hydroxylase after collagen is synthesized.
Natural collagen has a stable triple helix structure. Studies have shown that collagen molecules lacking hydroxyproline do not form stable triple helical structures at physiological temperatures, and that the important structural sequences of collagen often contain hydroxyproline. Collagen containing hydroxyproline is structurally closer to human collagen, has excellent biological activity, and is an advanced biological material. Therefore, the collagen hydroxylation level is improved, and the collagen which is more similar to the natural higher structure is obtained, so that the collagen has great market potential and prospect.
Pichia pastoris is often used for expression of recombinant human collagen due to the characteristics of high fermentation expression yield, simple culture medium and the like. However, in the prior art, the expression of recombinant human collagen is usually controlled by an AOX promoter in a Pichia pastoris expression system, the method cannot realize the separate regulation and control of the two proteins of human collagen and human hydroxylase, and the hydroxyproline in the recombinant human collagen obtained by the method is relatively low, so that the market development requirement of the collagen is difficult to meet.
Disclosure of Invention
The invention provides a method for improving the hydroxylation rate of recombinant human collagen aiming at the problem of low hydroxylation rate of collagen in a pichia pastoris expression system.
In order to achieve the above purpose, the invention adopts the following technical scheme: a method for increasing the rate of hydroxylation of recombinant human collagen comprising the steps of:
step one, constructing recombinant human collagen expression plasmids;
Step two, constructing a human hydroxylase expression plasmid, which comprises constructing a novel THI11 double-promoter vector and connecting a human hydroxylase expression sequence;
Step three, converting the recombinant human collagen expression plasmid and the human hydroxylase expression plasmid into pichia pastoris and screening to obtain recombinant strains capable of co-expressing the human collagen and the human hydroxylase;
and step four, the recombinant strain is induced and cultured by methanol and vitamin B1, and after the recombinant human collagen is purified, the content of proline and hydroxyproline amino acid in the recombinant human collagen is detected by a liquid phase method.
Preferably, the method for constructing the recombinant human collagen expression plasmid in the first step comprises the following steps: and selecting a full-length human III-type collagen alpha 1 chain nucleotide sequence capable of encoding the N-free short-end peptide, optimizing according to yeast codon preference, synthesizing a required recombinant human collagen DNA sequence, and connecting the sequence to a pichia pastoris expression plasmid to form the recombinant human collagen expression plasmid.
Preferably, the recombinant human collagen expression plasmid in the first step uses an AOX promoter.
Preferably, the method for constructing the THI11 double promoter vector in the second step is as follows: the THI11 promoter sequence was synthesized and ligated to the red yeast expression plasmid by restriction enzymes and T4 ligase to form a THI11 double promoter plasmid.
Preferably, in the method for constructing the THI11 double-promoter vector, the pichia pastoris expression plasmid is pPIC3.5K, pPIC9K, pPICZ alpha or pGAPZ alpha.
Preferably, the method for constructing the human hydroxylase expression plasmid in the second step comprises the following steps: optimizing according to alpha and beta subunit protein sequences of the human hydroxylase and preference of yeast codons, synthesizing required DNA fragments, and sequentially connecting the DNA fragments to a THI11 double-promoter vector to form the human hydroxylase expression plasmid.
Preferably, the signal peptide on the beta subunit protein sequence is replaced with an αmf signal peptide.
Preferably, the pichia pastoris in the third step is an X-33 strain, a GS115 strain or an SMD1168 strain.
Preferably, the content of vitamin B1 in the induction culture in the step four is 0-100 mug/L.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) The invention constructs a pichia pastoris expression strain, the strain is controlled by an AOX promoter to express human collagen and controlled by a THI11 promoter to express human hydroxylase, and the independent expression control of the human collagen and the human hydroxylase is realized by controlling the addition of methanol and vitamin B1, namely, the two proteins can be expressed together and regulated respectively.
(2) According to the invention, a pichia pastoris expression system is used for constructing a human collagen expression vector controlled by an AOX promoter and a human hydroxylase expression vector controlled by a THI11 promoter double promoter, and in the process of inducing expression by a recombinant strain, the addition amount of vitamin B1 of the recombinant strain under the culture condition of methanol is controlled to regulate and control hydroxylase expression, so that the occupation ratio of hydroxyproline in the recombinant human collagen is improved, and the hydroxylation rate of the recombinant human collagen is remarkably improved.
(3) The recombinant human collagen with the whole length is produced and obtained by the method, and the recombinant human collagen can resist the digestion of gastric protein and has good stability. In addition, the human collagen obtained by the method has no pyrogen problem, and the immunogenicity is effectively avoided.
Drawings
In order to more clearly illustrate the technical solution of the embodiments of the present invention, the following description of the embodiments will briefly describe the drawings that are required to be used in the description:
FIG. 1 is a schematic diagram of THI11 double promoter construction;
FIG. 2 is a graph showing the results of pepsin resistance test of recombinant collagen.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be rendered by reference to the appended drawings and examples.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein, and therefore the present invention is not limited to the specific embodiments of the disclosure that follow.
The BMGY medium composition was 1wt% yeast extract, 2wt% peptone, 1.34wt% YNB, 4X 10 -5 wt% biotin, 1wt% glycerol.
The BMMY medium composition was 1wt% yeast extract, 2wt% peptone, 1.34wt% YNB, 4X 10 -5 wt% biotin, 0.5wt% methanol.
Example 1
The invention is further described below, a method for increasing the rate of hydroxylation of recombinant human collagen, comprising the steps of:
step one, selecting a human III type collagen sequence, wherein the specific sequence is NCBI Reference Sequence NP-000081.2, and the sequence comprises a full-length human III type collagen alpha 1 chain which does not contain an N short-end peptide and contains a C-segment propeptide;
The selected protein sequence is optimized according to the preference of yeast codons, and the required recombinant human collagen DNA sequence is synthesized. The sequence of the synthesized DNA fragment is connected to a pMD-18T vector to form a recombinant plasmid, the recombinant plasmid is transformed into DH5 alpha for cloning, a monoclonal plasmid is extracted, two restriction enzymes EcoRI and NotI are used for double digestion, 0.8% agarose gel electrophoresis is used, and the target gene is recovered after gel cutting. The target gene is connected to pPICZ alpha A yeast expression vector to form recombinant expression plasmid of human collagen.
And step two, synthesizing a THI11 promoter sequence. The 5 'end of the THI11 promoter sequence sequentially contains SacI and AvrII restriction enzyme sites, the 3' end sequentially contains AvrII and BamHI restriction enzyme sites, the synthetic DNA fragment sequence is connected to a pMD-18T vector to form a pMD-18T recombinant plasmid, the recombinant plasmid is transformed into DH5 alpha for cloning, a monoclonal plasmid is extracted, the pMD-18T recombinant plasmid is subjected to single restriction enzyme avrII, 1.0% agarose gel electrophoresis is used for electrophoresis, and the THI11 promoter fragment is recovered by cutting gel and connected to a pPICZ alpha A plasmid to obtain the AOX-THI11 double promoter plasmid. The pMD-18T recombinant plasmid was then digested simultaneously with restriction enzymes SacI and BamHI, and the THI11 promoter fragment was recovered by excision of the gel. The recovered THI11 promoter fragment was substituted into the large disc AOX-THI11 double promoter plasmid to form a THI11 double promoter plasmid, and the synthesis process is shown in FIG. 1.
The sequences of the alpha and beta subunits of human hydroxylase are selected, with specific amino acid sequences referenced NCBI Reference Sequence:NP-001017962.1 and NCBI Reference Sequence:NP-000909.2. The signal peptide of the Beta subunit is replaced by an alpha MF signal peptide and optimized according to the preference of yeast codons, wherein the 5 'end of the Beta subunit DNA fragment contains Not I, an enzyme cutting site and a sequence capable of encoding the alpha MF signal peptide, and the 3' end contains an Age I enzyme cutting site. The 5 '-end of the alpha subunit DNA fragment contains BamHI cleavage site, and the 3' -end contains AOX1 terminator sequence and EcoRI cleavage site.
The synthetic DNA fragment sequence is connected to a pMD-18T vector to form a recombinant plasmid, the recombinant plasmid is transformed into DH5 alpha for cloning, a monoclonal plasmid is extracted, bamHI, ecoRI, notI and AgeI are used for respectively carrying out enzyme digestion on the alpha and beta subunit DNA sequences of the human hydroxylase, 1% agarose gel electrophoresis is used for carrying out gel cutting, and the target gene is recovered. The target gene is connected to the constructed THI11 double promoter plasmid vector before and after to form the recombinant expression plasmid of the human hydroxylase.
And thirdly, carrying out linearization treatment on the recombinant expression plasmid of the human collagen, converting the recombinant expression plasmid into Pichia pastoris X-33 in an electrotransformation mode, immediately adding sorbitol after conversion, uniformly mixing, coating the mixture on a YPD plate containing G418, culturing until colonies appear, and screening out positive clones.
Selecting 5 positive clones, respectively inoculating the positive clones into BMGY culture medium, culturing at 30 ℃ and 300r/min until the OD600 value is 4.0-6.0, centrifugally collecting thalli, then re-suspending cells by using the BMMY culture medium until the OD600 value is 1.0, fermenting and culturing at 30 ℃ and 300r/min, supplementing methanol every 12h, wherein the final concentration of the methanol is 0.5wt%, centrifugally collecting thalli after 96h of induced expression, and detecting the expression quantity of human collagen by SDS-PAGE analysis after ultrasonic crushing. Screening out the recombinant with highest expression of human collagen.
And (3) carrying out linearization treatment on the recombinant expression plasmid of the human hydroxylase, then converting the recombinant expression plasmid into the recombinant with high expression of the human collagen by an electrotransformation mode, immediately adding sorbitol after conversion, uniformly mixing, coating the mixture on a ZYPD flat plate containing G418, culturing until colonies appear, and screening out positive clones.
5 Positive clones are selected and respectively inoculated into BMGY culture medium, cultured at 30 ℃ and 300r/min until the OD600 value is 2.0-6.0, bacterial cells are centrifugally collected, the BMGY culture medium is added to enable the cell to reach the OD600 value of 1.0, fermentation culture is carried out at 30 ℃ and 300r/min, glycerol is added every 12 hours, the final concentration of the glycerol is 1wt%, after 96 hours of induced expression, bacterial cells are centrifugally collected, after ultrasonic crushing, the expression quantity of the human hydroxylase is detected by SDS-PAGE analysis, and recombinants with high expression of the human hydroxylase are selected.
Inoculating the obtained recombinants into a BMGY culture medium, culturing at 30 ℃ for 300r/min until the OD600 value is 4.0-6.0, centrifugally collecting thalli, adding the BMMY culture medium to enable the cell to reach the OD600 value of 1.0, fermenting and culturing at 30 ℃ for 300r/min, supplementing methanol and VB1 every 12 hours, wherein the final concentration of the methanol is 0.5wt%, the final concentration of the VB1 is 25 mug/L, and centrifugally collecting thalli after induced expression for 96 hours.
Taking a proper amount of thalli according to the following weight ratio of 1: and (3) adding the pre-cooled lysate into the mixture according to the proportion of 10, and then re-suspending the thalli in the lysate, and carrying out ice bath for 20min. The ultrasonic breaker probe is placed at 1cm below the liquid surface, ultrasonic breaking is carried out for 30min under the ice bath condition, and the supernatant is collected by centrifugation.
Because pPICZ alpha A contains His tag, the separation and purification of recombinant protein can be carried out by selecting an affinity chromatographic column nickel (Ni) column, and the specific steps are as follows: the supernatant was filtered, transferred to an equilibrated Ni Sepharose ™ HP chromatography column, and recombinant human collagen was eluted from the column with 250mmol/L imidazole eluent. The recombinant human collagen with the purity of 90% is obtained by SDS-PAGE analysis.
Taking a purified sample, desalting and concentrating the sample by using an ultrafiltration tube, and detecting the content of proline and hydroxyproline by adopting a liquid phase method, wherein the specific method comprises the following steps of:
Sample treatment: weighing a sample, dissolving in 6mol/L HCl solution, filling nitrogen, then digesting for 22 hours at 110 ℃, removing acid by nitrogen after digestion, filtering, fixing the volume in a 100mL volumetric flask, and diluting by proper times to obtain the product. Taking 10 mu L of standard substance solution to be measured in a derivative tube, adding 70 mu L of boric acid buffer solution, mixing with vortex, adding 20 mu L of derivatization reagent, standing for 1min after vortex mixing, sealing the derivative tube with a sealing film, and placing in a metal bath at 55 ℃ for heat preservation for 10min. The detection is carried out by high performance liquid chromatography, and the specific detection conditions are as follows:
Chromatographic column: inertsil ODS-SP, 4.6mm.times.250 mm,5 μm; at the fluorescence detection wavelength: excitation wavelength 250nm, emission wavelength 395nm; sample injection amount: 5. Mu.L; column temperature: 37 ℃; flow rate: 1mL/min; mobile phase a: 11.48g of anhydrous sodium acetate, 1.72g of triethylamine and EDTA:1mg, dissolved in 1L of water, adjusted to pH 4.95 with phosphoric acid; mobile phase B:60% acetonitrile in water.
And (3) calculating results: and drawing a standard curve by taking the concentration of the standard substance of the proline and the hydroxyproline as an abscissa and the liquid phase peak area as a coordinate, calculating the content of the proline and the hydroxyproline according to the corresponding liquid phase peak area of the sample, and calculating the proportion of the hydroxyproline in the total proline. The hydroxyproline was calculated to be 46.6% of the total proline.
Example 2
This embodiment differs from embodiment 1 in that: inoculating the obtained recombinants into a BMGY culture medium, culturing at 30 ℃ for 300r/min until the OD600 value is 4.0-6.0, centrifugally collecting thalli, adding the BMMY culture medium to enable the cell to reach the OD600 value of 1.0, fermenting and culturing at 30 ℃ for 300r/min, supplementing methanol and VB1 every 12 hours, wherein the final concentration of the methanol is 0.5wt%, the final concentration of the VB1 is 0 mug/L, and centrifugally collecting thalli after induced expression for 96 hours.
The cells were sonicated and purified using a Ni Sepharose ™ HP column. And (3) taking a purified sample, desalting and concentrating the sample by using an ultrafiltration tube, detecting the content of the proline and the hydroxyproline by using a liquid phase method, and calculating the proportion of the hydroxyproline in the total proline to be 54.5%.
Example 3
This embodiment differs from embodiment 1 in that: inoculating the obtained recombinants into a BMGY culture medium, culturing at 30 ℃ for 300r/min until the OD600 value is 4.0-6.0, centrifugally collecting thalli, adding the BMMY culture medium to enable the cell to reach the OD600 value of 1.0, fermenting and culturing at 30 ℃ for 300r/min, supplementing methanol and VB1 every 12 hours, wherein the final concentration of the methanol is 0.5wt%, the final concentration of the VB1 is 50 mug/L, and centrifugally collecting thalli after induced expression for 96 hours.
The cells were sonicated and purified using a Ni Sepharose ™ HP column. And (3) taking a purified sample, desalting and concentrating the sample by using an ultrafiltration tube, detecting the content of the proline and the hydroxyproline by using a liquid phase method, and calculating the ratio of the hydroxyproline to the total proline to be 38.2%.
Example 4
This embodiment differs from embodiment 1 in that: inoculating the obtained recombinants into a BMGY culture medium, culturing at 30 ℃ for 300r/min until the OD600 value is 4.0-6.0, centrifugally collecting thalli, adding the BMMY culture medium to enable the cell to reach the OD600 value of 1.0, fermenting and culturing at 30 ℃ for 300r/min, supplementing methanol and VB1 every 12 hours, wherein the final concentration of the methanol is 0.5wt%, the final concentration of the VB1 is 75 mug/L, and centrifugally collecting thalli after induced expression for 96 hours.
The cells were sonicated and purified using a Ni Sepharose ™ HP column. And (3) taking a purified sample, desalting and concentrating the sample by using an ultrafiltration tube, detecting the content of the proline and the hydroxyproline by using a liquid phase method, and calculating the ratio of the hydroxyproline to the total proline to be 31.6%.
Example 5
This embodiment differs from embodiment 1 in that: inoculating the obtained recombinants into a BMGY culture medium, culturing at 30 ℃ for 300r/min until the OD600 value is 4.0-6.0, centrifugally collecting thalli, adding the BMMY culture medium to enable the cells to reach the OD600 value of 1.0, fermenting and culturing at 30 ℃ for 300r/min, supplementing methanol and VB1 every 12h, wherein the final concentration of the methanol is 0.5wt%, the final concentration of the VB1 is 100 mu g/L, and centrifugally collecting the thalli after induced expression for 96 h.
The cells were sonicated and purified using a Ni Sepharose ™ HP column. And (3) taking a purified sample, desalting and concentrating the sample by using an ultrafiltration tube, detecting the content of the proline and the hydroxyproline by using a liquid phase method, and calculating the proportion of the hydroxyproline in the total proline to be 20.9%.
As can be seen from examples 1-5, the present invention constructs a Pichia expression strain that expresses human collagen under the control of the AOX promoter and human hydroxylase under the control of the THI11 promoter. The expression strain is used for regulating and controlling the expression of hydroxylase by controlling the addition amount of vitamin B1 under the culture condition of containing methanol, so that the ratio of hydroxyproline in the recombinant human collagen is increased to 54.5 percent, and the hydroxylation rate of the recombinant human collagen is close to that of natural human collagen.
It should be noted that, natural collagen has a stable triple helix structure, so that it has a strong resistance to common proteases, and is not easy to degrade. Thus, the present invention also examined the recombinant obtained in the examples for pepsin tolerance of recombinant human collagen obtained under the following test conditions: the recombinants obtained in the example are inoculated into BMGY culture medium, cultured at 30 ℃ and 300r/min until the OD600 value is 4.0-6.0, bacterial cells are collected by centrifugation, the BMMY culture medium is added to enable the cells to reach the OD600 value of 1.0, fermentation culture is carried out at 30 ℃ and 300r/min, methanol is added every 12 hours, the final concentration of the methanol is 0.5wt%, and after 96 hours of induced expression, bacterial cells are collected by centrifugation. After the cells were sonicated, they were purified by Ni Sepharose ™ HP chromatography. Taking a purified sample, adding 0.2mg/ml pepsin, reacting for 1 hour in a constant temperature incubator at 37 ℃, and then adding 100mM NaOH solution to terminate the pepsin digestion reaction; detection was performed by SDS-PAGE electrophoresis. As shown in FIG. 2, the collagen band with the size of 180KD is obtained after fermentation and purification, and the protein band is obtained after digestion by pepsin, but the size of the collagen band is obviously reduced, which indicates that the collagen forms a triple helix structure and can resist digestion by pepsin, and the section part of C which does not form the triple helix is digested by pepsin, so that the protein size is reduced.
The present invention is not limited to the above-mentioned embodiments, and any equivalent embodiments which can be changed or modified by the technical content disclosed above can be applied to other fields, but any simple modification and equivalent changes to the above-mentioned embodiments according to the technical substance of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (9)
1. A method for increasing the rate of hydroxylation of recombinant human collagen comprising the steps of:
step one, constructing recombinant human collagen expression plasmids;
Step two, constructing a human hydroxylase expression plasmid, which comprises constructing a novel THI11 double-promoter vector and connecting a human hydroxylase expression sequence;
Step three, converting the recombinant human collagen expression plasmid and the human hydroxylase expression plasmid into pichia pastoris and screening to obtain recombinant strains capable of co-expressing the human collagen and the human hydroxylase;
and step four, the recombinant strain is induced and cultured by methanol and vitamin B1, and after the recombinant human collagen is purified, the content of proline and hydroxyproline amino acid in the recombinant human collagen is detected by a liquid phase method.
2. The method for increasing hydroxylation rate of recombinant human collagen according to claim 1, wherein the method for constructing the recombinant human collagen expression plasmid in the first step comprises the steps of: and selecting a full-length human III-type collagen alpha 1 chain nucleotide sequence capable of encoding the N-free short-end peptide, optimizing according to yeast codon preference, synthesizing a required recombinant human collagen DNA sequence, and connecting the sequence to a pichia pastoris expression plasmid to form the recombinant human collagen expression plasmid.
3. The method according to claim 1, wherein the AOX promoter is used in the recombinant human collagen expression plasmid in the first step.
4. The method for increasing hydroxylation rate of recombinant human collagen according to claim 1, wherein the method for constructing the THI11 dual promoter vector in the second step comprises the steps of: the THI11 promoter sequence is synthesized and is connected to the pichia pastoris expression plasmid through restriction enzyme and T4 ligase to form the THI11 double-promoter expression vector.
5. The method for increasing hydroxylation rate of recombinant human collagen according to claim 4, wherein the pichia pastoris expression plasmid in the method for constructing the THI11 double promoter vector is ppic3.5K, ppic9K, pPICZ a or pGAPZ a.
6. The method for increasing hydroxylation rate of recombinant human collagen according to claim 5, wherein the method for constructing the human hydroxylase expression plasmid in the second step comprises the steps of: the corresponding DNA fragments are synthesized by optimizing the alpha subunit, beta subunit protein sequence and yeast codon preference of the human hydroxylase, and are sequentially connected to a THI11 double-promoter expression vector through restriction enzymes and T4 ligase to form the human hydroxylase expression plasmid.
7. The method of claim 6, wherein the signal peptide on the beta subunit protein sequence is replaced with an alpha MF signal peptide.
8. The method of claim 1, wherein the pichia pastoris in step three is X-33 strain, GS115 strain or SMD1168 strain.
9. The method for increasing the hydroxylation rate of recombinant human collagen according to claim 1, wherein the content of vitamin B1 in the induction culture in the fourth step is 0-100 μg/L.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011123811A2 (en) * | 2010-04-02 | 2011-10-06 | The Board Of Trustees Of The Leland Stanford Junior University | Production of post-translationally hydroxylated recombinant proteins in bacteria |
| CN111374313A (en) * | 2020-04-24 | 2020-07-07 | 天赋能(天津)健康产业集团股份有限公司 | Liquid meal replacement capable of controlling weight and preparation method thereof |
| CN114957449A (en) * | 2022-02-24 | 2022-08-30 | 吉林省国大生物工程有限公司 | Preparation method of recombinant human type III collagen |
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| CN116555320A (en) * | 2023-07-06 | 2023-08-08 | 天津辅元生物医药科技有限公司 | Recombinant human-derived III-type triple helix collagen engineering bacterium, and construction method and application thereof |
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| WO2011123811A2 (en) * | 2010-04-02 | 2011-10-06 | The Board Of Trustees Of The Leland Stanford Junior University | Production of post-translationally hydroxylated recombinant proteins in bacteria |
| CN111374313A (en) * | 2020-04-24 | 2020-07-07 | 天赋能(天津)健康产业集团股份有限公司 | Liquid meal replacement capable of controlling weight and preparation method thereof |
| CN115704040A (en) * | 2021-08-06 | 2023-02-17 | 华东理工大学 | Transcription regulation system based on CRISPRi and CRISPRa, its establishment method and application |
| CN114957449A (en) * | 2022-02-24 | 2022-08-30 | 吉林省国大生物工程有限公司 | Preparation method of recombinant human type III collagen |
| CN116555320A (en) * | 2023-07-06 | 2023-08-08 | 天津辅元生物医药科技有限公司 | Recombinant human-derived III-type triple helix collagen engineering bacterium, and construction method and application thereof |
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| 尼尔斯•兰德斯: "The vitamin-sensitive promoter PTHI11 enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris", BIOTECHNOLOGY AND BIOENGINEERING, vol. 113, no. 12, 27 June 2016 (2016-06-27), pages 1 * |
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