CN117700530A

CN117700530A - Purification production method of recombinant IV-type humanized collagen containing dopa

Info

Publication number: CN117700530A
Application number: CN202311685483.9A
Authority: CN
Inventors: 郝东; 侯增淼; 赵硕文; 魏文培; 周浩; 李敏; 乔莎; 张立峰
Original assignee: Xi'an Denos Medical Technology Co ltd
Current assignee: Xi'an Denos Medical Technology Co ltd
Priority date: 2023-12-08
Filing date: 2023-12-08
Publication date: 2024-03-15

Abstract

The invention discloses a purification production method of recombinant IV type humanized collagen containing dopa, which comprises the steps of carrying out solid-liquid separation, filtration clarification and ultrafiltration concentration on pichia pastoris engineering bacteria fermentation liquor, then carrying out enzymatic reaction and composite cation filler capture, and carrying out immersion washing, ultrafiltration concentration, strong anion chromatography and freeze drying to obtain the recombinant IV type humanized collagen with purity of more than 95%, dopa content of more than 0.3 and endotoxin content of less than 1 EU/mg.

Description

Purification production method of recombinant IV-type humanized collagen containing dopa

Technical Field

The invention belongs to the technical field of protein engineering, and relates to an industrialized separation and purification and dopa modification method of a recombinant IV-type humanized collagen crude product feed liquid obtained by fermentation.

Background

Collagen (collagen) is the most abundant protein in the body, accounting for 25% -33% of the total protein, and is widely distributed in various tissues and organs of the body, such as skin, bone, cornea, blood vessels, etc., especially in skin and connective tissues of human body, and contains a large amount of collagen. Collagen, which is a connective tissue adhesive substance, plays an important role in maintaining normal physiological functions of cells, tissues and organs. The recombinant collagen has the advantages of low toxicity, low antigenicity and low immunity, can guide cell regeneration and is compatible with human body, and can be widely applied to industries such as biological medicine, cosmetics, food and the like.

3, 4-Dihydroxyphenylalanine (DOPA) is a structural component of mussel mucin, and is formed by utilizing the modification effect of tyrosinase on tyrosine residues. DOPA residues are crosslinked with each other due to oxidation reaction, so that mussel mucin and surface fibers form strong and durable adhesive bonding, the bonding capacity and the service life are not affected by water, and the adhesive effect has no toxicity and immunogenicity to people, and the adhesive has wide application prospect. The amino acid composition of the type IV collagen contains tyrosine residues, so that the DOPA modification reprocessing is carried out on an expression product derived from the natural type IV collagen sequence structure to obtain the recombinant type IV collagen containing DOPA residues, and the method has important significance in the aspect of expanding the application of the type IV collagen.

There are few reports on the technology of preparing recombinant type IV collagen pure product containing DOPA. Existing techniques for dopa modification in the expression of recombinant proteins from different sources or after expression (e.g., CN115819627A, CN115894655 a) generally require the use of chromatographic columns for the purification process to separate the modified recombinant proteins, which not only results in complex processes, but also results in low purity of the final product.

Disclosure of Invention

The invention aims to provide a purification production method of recombinant IV-type humanized collagen containing dopa, which is used for purifying feed liquid (namely fermentation liquid) obtained by fermenting pichia pastoris engineering bacteria to efficiently obtain a recombinant protein pure product with dopa groups.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the purification production method of the collagen comprises the following steps:

after the fermentation of the pichia pastoris engineering bacteria for expressing the recombinant type IV collagen is finished, carrying out fermentation liquor pretreatment to obtain an intermediate material liquid containing the recombinant type IV collagen; mixing the intermediate product feed liquid with phosphate, copper sulfate, tyrosinase and MMC composite cation exchange filler (which is washed by purified water in advance to remove impurities such as filler preservation reagent and the like), regulating pH to 7.4-7.8 by alkali (such as sodium hydroxide and disodium hydrogen phosphate), performing enzymatic reaction in a liquid phase by using the tyrosinase, regulating a stock solution obtained by the enzymatic reaction to be acidic by using acid or acid solution after the enzymatic reaction is finished, replacing recombinant IV type collagen modified by the enzymatic reaction (namely recombinant IV type collagen containing dopa) on the MMC composite cation exchange filler, and separating the MMC composite cation exchange filler by using a screen; eluting the MMC composite cation exchange filler trapped on the sieve screen by using an acid solution to remove residual tyrosinase and other impurities, eluting the recombinant IV type collagen subjected to enzymatic reaction modification, which is replaced on the MMC composite cation exchange filler, by using a buffer solution, and performing aftertreatment on the obtained eluent to obtain the recombinant IV type collagen containing dopa.

Preferably, the pretreatment specifically includes the following steps: and (3) after fermentation, placing the fermentation tank to obtain fermentation liquor containing the pichia pastoris engineering bacteria and recombinant IV-type collagen obtained by expression of the pichia pastoris engineering bacteria, and sequentially carrying out solid-liquid separation (for example, removing sediment and collecting supernatant after centrifuging by a high-speed centrifuge), filtering and clarifying (for example, filtering the supernatant obtained by the centrifugation by a membrane system, wherein the membrane system is a 0.22 or 0.45 mu m hollow fiber membrane filtration system) and ultrafiltration concentration (the retentate obtained by ultrafiltration is the intermediate feed liquid).

Preferably, the recombinant type IV collagen expressed by the pichia pastoris engineering bacteria has a sequence structure including one or more fusion sequence units (for example, recombinant type IV humanized collagen having an amino acid sequence shown in seq id No.1 and including a plurality of identical fusion sequence units) composed of a sequence of a peptide fragment selected from the natural type IV collagen.

Preferably, the fermentation specifically comprises the following steps: culturing the pichia pastoris gene engineering bacteria as primary seeds in a fermentation tank (for example, 10L) (the culture temperature is 25-28 ℃), inoculating the cultured bacteria as secondary seeds in the fermentation tank (for example, 100L) for culture (the culture temperature is 25-28 ℃), and carrying out secretion expression of the recombinant IV type collagen in the culture process; and discharging fermentation liquor (i.e. discharging the tank) after fermentation.

Preferably, in the pretreatment, an ultrafiltration concentration adopts a roll-type filter membrane ultrafiltration system of less than or equal to 6KD, and the ultrafiltration is carried out until the conductivity of the trapped fluid is less than 0.5mS/cm (preventing the efficiency of the enzymatic reaction from being influenced by the excessive conductivity of the trapped fluid).

Preferably, the phosphate is disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and the addition amount of the copper sulfate, tyrosinase, MMC composite cation exchange filler, disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in the intermediate material liquid is 0.01-0.02 g/L (copper sulfate), 100-200U/L (tyrosinase), 25-30% (MMC composite cation exchange filler, volume ratio), 0.9-1.1 g/L (disodium hydrogen phosphate dodecahydrate) and 0.32-0.35 g/L (sodium dihydrogen phosphate dihydrate) in sequence.

Preferably, the specific conditions of the enzymatic reaction are: stirring at 22-28 deg.c (e.g. room temperature or normal temperature) for 3-4 hr at 100-200 rpm.

Preferably, the acid (the stock solution obtained by the above enzymatic reaction is regulated to be acidic) is one of hydrochloric acid, sulfuric acid, citric acid and glacial acetic acid, wherein the acid in the form of solid particles, such as citric acid, can avoid the operation risk caused by the irritation and volatility of other liquid acids or solutions thereof; the replacement specifically comprises the following steps: the stock solution obtained by the enzymatic reaction is stirred for 1 to 2 hours (during stirring, impurities such as protein expressed by host pichia pastoris per se can be desorbed from the surface of the filler, and recombinant IV type collagen modified by the enzymatic reaction can be bound to the surface of the filler, so that replacement is completed) after the pH of the stock solution obtained by the enzymatic reaction is regulated to 3.5 to 4.5 by using acid (such as citric acid) or acid solution.

Preferably, the acid solution adopted in leaching is acetic acid solution with pH of 3.5-4 or citric acid solution with pH of 3.5-4, and the leaching temperature is room temperature or normal temperature.

Preferably, the citric acid solution or acetic acid solution is used in the leaching in an amount of not less than 5 times the volume of the retained packing (i.e., MMC composite cation exchange packing retained on the screen).

Preferably, the specific conditions of the elution are: the corresponding filler (specifically, MMC composite cation exchange filler subjected to the replacement and leaching) combined with the recombinant IV type collagen subjected to the modification of the enzymatic reaction, which is formed after the replacement, is soaked in 10-15 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH of 6.5-7 for more than one time (for example, the soaking times are not less than 3 times and the using amount of the buffer solution is not less than the volume of entrapped filler each time), the soaking time is not less than 30 minutes each time (in order to ensure that the recombinant IV type collagen subjected to the modification of the enzymatic reaction is fully desorbed from the surface of the filler in the soaking process, the soaking time is too short, the protein is insufficiently desorbed, the yield is reduced), and the soaking temperature is room temperature or normal temperature.

Preferably, the replacement is completed and then is filtered by a screen mesh not smaller than 200 meshes, so that MMC composite cation exchange filler added in the intermediate product liquid is separated (trapped) as much as possible; and filtering the eluate (particularly, soaking in the eluate) through a screen mesh not smaller than 200 meshes, and collecting filtrate, thereby eliminating the need of eluting on a chromatographic column and reducing the process cost.

Preferably, the post-treatment specifically comprises the following steps: and performing ultrafiltration concentration on the eluent, and then performing freeze drying, or performing ultrafiltration concentration on the eluent, and then sequentially performing endotoxin removal and freeze drying. I.e. the retentate obtained by ultrafiltration in the post-treatment is directly lyophilized or lyophilized after endotoxin removal.

Preferably, in the post-treatment, an ultrafiltration concentration adopts a roll-type filter membrane ultrafiltration system with the thickness of less than or equal to 6KD, and the ultrafiltration is carried out until the conductivity of the trapped fluid is less than 0.1mS/cm.

Preferably, in the post-treatment, endotoxin removal is performed by using a strong anion chromatography column (e.g., QFF chromatography column, previously equilibrated with purified water to avoid the introduction of other impurities, and the column feed solution is collected for the above freeze-drying).

Preferably, the freeze-dried powder obtained by freeze-drying is a recombinant type IV collagen finished product (endotoxin content is less than 1 EU/mg) with the purity of more than 95 percent, the yield is more than 70 percent (the yield of fermentation liquor per liter is more than 0.8 g/L), and the dopa content is more than 0.3 percent.

The beneficial effects of the invention are as follows:

the purification production method of the collagen provided by the invention takes the fermentation liquor of the pichia pastoris genetically engineered bacteria as a raw material, mainly adopts enzymatic reaction and composite cationic filler to carry out dopa modification and capture on the recombinant IV type collagen induced to be expressed, and in the purification process of the invention, the pH is regulated to 7.4-7.8 by alkali before the enzymatic reaction, so that impurities in intermediate product liquid are fully combined with the filler, the purification in the enzymatic reaction is realized (and the enzymatic reaction is promoted, thereby improving the dopa modification effect), and then the impurity removal can be realized by utilizing acid solution to wash after the completion of the capture of the filler and the modified protein (without carrying out on a chromatographic column), so that the purification process is simple, and the purity of the finished protein is high, thereby being suitable for industrial production.

Furthermore, the pH value is regulated to 3.5-4.5 by using acid or acid solution after the enzymatic reaction, so that the modified protein (such as recombinant IV-type humanized collagen) is fully combined with the filler (and protein degradation is avoided), and the insufficient dissociation of impurities and the filler (insufficient combination of the recombinant IV-type humanized collagen modified by the enzymatic reaction and the filler) caused by too short stirring time is avoided by controlling the stirring time, thereby improving the yield of the finished product protein and the like.

Furthermore, the invention adopts a strong anion chromatographic column to remove endotoxin after ultrafiltration and concentration of eluent, so that the endotoxin content of the finished product protein can be reduced.

Drawings

FIG. 1 shows the result of the color reaction of a blue nitrotetrazolium chloride (NBT) dye solution; wherein, 1: example 1 centrifugation of supernatant samples after fermentation; 2: comparative example 1 stock samples after enzymatic reaction; 3: example 1 stock samples after enzymatic reaction.

FIG. 2 is a SDS-PAGE electrophoresis of samples of the recombinant type IV collagen purification process of example 1; wherein, lane 1: centrifuging the supernatant sample after fermentation; lane 2: a stock sample (6-fold dilution) after enzymatic reaction; lane 3: an acid solution leacheate sample; lane 4: an eluent sample; lane 5: protein markers; lanes 1 to 5 were spotted in an amount of 10. Mu.L.

Detailed Description

The invention will now be described in further detail with reference to the drawings and examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

Example 1

1. After the fermentation of the Pichia pastoris engineering bacteria is finished, the Pichia pastoris engineering bacteria are placed in a tank, the volume of fermentation broth is 57L, the fermentation broth is centrifuged at 4000rpm for 15min by a high-speed centrifuge (room temperature), the precipitate is discarded, the supernatant is collected, the volume of the supernatant is 36L, and the target protein (recombinant IV type humanized collagen without dopa modification, namely an intermediate product; fig. 2) crude product feed liquid is obtained by collecting the supernatant.

2. The supernatant collected in step 1 was clarified by filtration through a 0.45 μm hollow fiber filtration apparatus (room temperature), the retentate was diluted and filtered with 10L of purified water to recover the residual target protein, and the filtrate was collected to have a filtrate volume of 41L.

3. The filtrate collected in the step 2 is ultrafiltered and concentrated by a 6KD (selected according to the molecular weight of target protein) roll-type membrane ultrafiltration system (room temperature), and when purified water (dialyzed, the ion concentration in the trapped fluid is reduced) is added to the trapped fluid with the conductivity of 0.46mS/cm, the trapped fluid is collected, and the volume of the trapped fluid is 5L.

4. To the retentate collected in step 3, 5g of disodium hydrogen phosphate dodecahydrate (i.e., 1.0 g/L), 1.65g of sodium dihydrogen phosphate dihydrate (i.e., 0.33 g/L), 0.07g of copper sulfate (i.e., 0.01 g/L) and 0.02g of tyrosinase (i.e., 120U/L) designated as 30U/mg were added, stirred until completely dissolved, then 1.3L of MMC composite cation exchange filler was added at a volume ratio of 26%, pH was adjusted to 7.5 with sodium hydroxide, and stirred at 100rpm for 3.5 hours to perform an enzymatic reaction (room temperature).

5. Citric acid was added to the final stock solution (FIG. 2) obtained in the enzymatic reaction of step 4 to adjust the pH to 3.8, and stirring was continued at 100rpm for 1 hour (room temperature).

6. Filtering the feed liquid treated in the step 5 by using a 200-mesh screen, intercepting the MMC composite cation exchange filler on the screen to form a filter cake, leaching (room temperature) the filter cake by using 7L citric acid solution with pH of 3.7, and collecting the filler.

7. Immersing (at room temperature) the MMC composite cation exchange filler (i.e. the collected filler) treated in the step 6 in 1.5L

Finished product elution was performed in 10mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pH6.6 for 30min (FIG. 2), the eluate was collected by filtration through a 200 mesh screen (i.e., the filtrate was collected), the 1-eluting filler trapped on the screen was further subjected to the above soaking and filtration processes 2 times, and the 3-collected eluents were combined for a total of 4.4L.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water (dialyzing to reduce the ion concentration in the trapped fluid) until the conductivity of the trapped fluid reaches 91 mu S/cm, collecting 2.3L of the trapped fluid, collecting the column-penetrating liquid after the trapped fluid is subjected to column-penetrating (room temperature) chromatography column loading (well-balanced) by the purified water, and freeze-drying the obtained column-penetrating liquid to obtain 46.26g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquid is 0.811g/L, namely 71.6%.

9. Through electrophoresis detection, the purity of the freeze-dried powder sample is 97.1%; the content of dopa in the freeze-dried powder sample is 0.36 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

Example 2

1. And (3) placing the pichia pastoris engineering bacteria in a tank after fermentation is finished, centrifuging the fermentation liquid at a high-speed centrifuge (room temperature) for 15min at 4000rpm, discarding the precipitate, collecting the supernatant with a supernatant volume of 36L, and collecting the supernatant to obtain crude product feed liquid of target protein (recombinant IV-type humanized collagen without dopa modification, namely an intermediate product).

3. And (3) ultrafiltering and concentrating the filtrate collected in the step (2) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), and collecting the trapped fluid when the conductivity of the trapped fluid is 0.48mS/cm by adding purified water, wherein the volume of the trapped fluid is 5L.

4. To the retentate collected in step 3, 5.4g of disodium hydrogen phosphate dodecahydrate (i.e., 1.1 g/L), 1.74g of sodium dihydrogen phosphate dihydrate (i.e., 0.35 g/L), 0.09g of copper sulfate (i.e., 0.02 g/L) and 0.03g of tyrosinase (i.e., 180U/L) marked as 30U/mg were added, stirred until completely dissolved, then 1.5L of MMC composite cation exchange filler was added at a volume ratio of 30%, pH was adjusted to 7.7 with sodium hydroxide, and stirred at 100rpm for 4 hours to perform an enzymatic reaction (room temperature).

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 4.4, and stirring for 2 hours (room temperature) at 100 rpm.

6. Filtering the feed liquid treated in the step 5 by using a 200-mesh screen, intercepting the MMC composite cation exchange filler on the screen to form a filter cake, leaching the filter cake by using 7.6L citric acid solution with pH of 3.9 (room temperature), and collecting the filler.

The finished product is eluted in 15mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 6.9 for 30min, the eluent is filtered and collected by a 200-mesh screen (namely, filtrate is collected), the filling material which is trapped on the screen and subjected to 1 time of elution is subjected to the soaking and filtering processes for 2 times, and the total eluent collected for 3 times is 4.4L.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 98 mu S/cm, collecting 2.3L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 46.79g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquid is 0.821g/L, namely 73.2%.

9. Through electrophoresis detection, the purity of the freeze-dried powder sample is 97.3%; the content of dopa in the freeze-dried powder sample is 0.36 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

Example 3

3. And (3) ultrafiltering and concentrating the filtrate collected in the step (2) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), and collecting the trapped fluid when the conductivity of the trapped fluid is 0.42mS/cm by adding purified water, wherein the volume of the trapped fluid is 5L.

4. To the retentate collected in step 3, 4.6g of disodium hydrogen phosphate dodecahydrate (i.e., 0.9 g/L), 1.63g of sodium dihydrogen phosphate dihydrate (i.e., 0.33 g/L), 0.05g of copper sulfate (i.e., 0.01 g/L) and 0.02g of tyrosinase (i.e., 120U/L) marked as 30U/mg were added, stirred until completely dissolved, then 1.3L of MMC composite cation exchange filler was added at a volume ratio of 26%, pH was adjusted to 7.4 with sodium hydroxide, and stirred at 100rpm for 3 hours to perform an enzymatic reaction (room temperature).

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 3.5, and stirring for 1h (room temperature) at 100 rpm.

6. Filtering the feed liquid treated in the step 5 by using a 200-mesh screen, intercepting the MMC composite cation exchange filler on the screen to form a filter cake, leaching the filter cake by using 6.6L citric acid solution with pH of 3.5 (room temperature), and collecting the filler.

7. Immersing (at room temperature) the MMC composite cation exchange filler (i.e. the collected filler) treated in the step 6 in 1.3L

Finished product elution is carried out in 10mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 6.5 for 30min, the eluent is filtered and collected through a 200-mesh screen (namely filtrate is collected), the filling material which is trapped on the screen and subjected to 1 time of elution is subjected to the soaking and filtering processes for 2 times, and the eluent collected for 3 times is combined for 3.8L in total.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 89 mu S/cm, collecting 2.1L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 46.53g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquid is 0.802g/L, namely 70.8%.

9. Detecting by electrophoresis, wherein the purity of the freeze-dried powder sample is 96.9%; the content of dopa in the freeze-dried powder sample is 0.35 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

Example 4

8. And 5, ultrafiltering and concentrating the eluent collected in the step 7 by a 6KD roll-type filter membrane ultrafiltration system (room temperature), when purified water is added to reach the conductivity of 89 mu S/cm of the trapped fluid, collecting 2.1L of the trapped fluid, and freeze-drying to obtain 48.31g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquor is 0.833g/L, namely, the yield is 74.2%.

9. Through electrophoresis detection, the purity of the freeze-dried powder sample is 97.2%; the content of dopa in the freeze-dried powder sample is 0.32 percent by the content measurement of dopa; endotoxin-free control can be performed prior to use of the finished protein.

In the above examples, the Pichia pastoris engineering bacteria are Pichia pastoris engineering bacteria capable of expressing recombinant type IV collagen, the engineering bacteria are obtained by constructing Pichia pastoris GS115 strain as host bacteria, the amino acid sequence of the expressed recombinant type IV collagen is shown as SEQ ID No.1 in Table 1 (refer to human type IV collagen GenBank: AAI51221.1, and the recombinant type IV humanized collagen is obtained by selecting and combining peptide fragments into a sequence unit with the length of 90 aa. Determining a gene sequence according to a designed amino acid sequence and combining preference of pichia pastoris to codons, simultaneously adding Xho I restriction enzyme cutting sites CTCGAG and KEX2 enzyme cutting sites AAAAGA (N-terminal alpha-factor signal peptide can be cut off from the sites when target protein is secreted outside cells) at the 5 'end of the gene sequence, and adding a stop codon and Not I restriction enzyme cutting sites GCGGCCGC at the 3' end in order to complete subsequent molecular operation; and (3) carrying out total gene synthesis on the designed nucleotide sequence (namely SEQ. ID. NO.2 in Table 1) to obtain a synthetic sequence fragment containing the target gene.

Construction of the expression vector: the synthesized sequence fragment was double digested with Xho I and Not I and ligated into the Pichia pastoris vector pPIC9K (Invitrogen) after the same double digestion.

Transformation of expression vectors and screening of positive recombinant Pichia pastoris (i.e., pichia pastoris engineering bacteria) are referred to in the Pichia expression handbook.

TABLE 1 sequence design results

In the above examples, pichia pastoris engineering bacteria are used as primary seeds to be cultured in a 10L fermentation tank for 18 hours, the cultured bacteria are used as secondary seeds to be inoculated in a 100L fermentation tank, secretion expression of recombinant IV type collagen is carried out in the culture process, and the fermentation liquid can be obtained after the fermentation is finished; wherein, the culture medium of the first-class seeds, the specific culture conditions, the culture medium of the second-class seeds and the specific culture conditions refer to the Pichia pastoris fermentation handbook.

In the above embodiments, the electrical conductivity of ultrafiltration concentration in the step 3 provides conditions for subsequent enzymatic reactions, and ensures that the enzymatic reaction process is performed efficiently; in the step 4, partial impurity proteins (mainly expressed by host bacteria) are combined with MMC composite cation exchange filler, and tyrosinase and target proteins are not combined with the MMC composite cation exchange filler under the pH condition regulated by sodium hydroxide, so that the purity of the target proteins in a solution system is improved, the contact area of tyrosinase and a substrate is increased, and the enzymatic reaction efficiency is improved; in the step 5, the target protein modified by enzymatic reaction under the pH condition regulated by citric acid is combined with MMC composite cation exchange filler, so as to realize the capture of the recombinant IV-type collagen containing dopa; in the step 6, enough citric acid solution is used for leaching to remove tyrosinase and other impurities which are not combined with MMC composite cation filler (mainly refers to various substances which are not combined with the filler and are generated in the fermentation process); in the step 7, filling is soaked in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution to elute target protein modified by enzymatic reaction, so as to obtain recombinant IV type collagen pure solution containing dopa; and (8) after ultrafiltration and concentration, collecting the column-penetrating feed liquid through a QFF chromatographic flow-through mode to remove endotoxin as clean as possible, or directly obtaining freeze-dried powder through freeze drying without the endotoxin removal treatment. The effect of the purification process and parameters can be seen in the following comparative examples.

Comparative example 1

1. And (3) placing the pichia pastoris engineering bacteria in a tank after fermentation is finished, centrifuging the fermentation liquid at a high-speed centrifuge (room temperature) for 15min at 4000rpm, discarding the precipitate, collecting the supernatant with a supernatant volume of 36.7L, and collecting the supernatant to obtain crude product feed liquid of target protein (recombinant IV-type humanized collagen without dopa modification, namely an intermediate product).

2. The supernatant collected in step 1 was clarified by filtration through a 0.45 μm hollow fiber filtration apparatus (room temperature), the retentate was diluted and filtered with 10L of purified water to recover the residual target protein, and the filtrate was collected to have a filtrate volume of 41.4L.

3. And (3) ultrafiltering and concentrating the filtrate collected in the step (2) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), and collecting the trapped fluid when the conductivity of the trapped fluid is 0.42mS/cm by adding purified water, wherein the trapped fluid volume is 5.1L.

4. To the retentate collected in step 3 were added 5.1g of disodium hydrogen phosphate dodecahydrate (i.e., 1.0 g/L), 1.67g of sodium dihydrogen phosphate dihydrate (i.e., 0.33 g/L), 0.07g of copper sulfate (i.e., 0.01 g/L) and 0.0205g of tyrosinase (i.e., 120U/L) labeled 30U/mg, stirred until completely dissolved, pH was adjusted to 7.5 with sodium hydroxide, and stirred at 100rpm for 3.5 hours for enzymatic reaction (room temperature).

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 3.8, then adding 1.33L MMC composite cation exchange filler according to the volume ratio of 26%, and continuously stirring at 100rpm for 1h (room temperature).

Finished product elution is carried out in 10mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 6.6 for 30min, the eluent is filtered and collected through a 200-mesh screen (namely filtrate is collected), the filling material which is trapped on the screen and subjected to 1 time of elution is subjected to the soaking and filtering processes for 2 times, and the total eluent collected for 3 times is combined for 4.5L.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 89 mu S/cm, collecting 2.4L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 46.74g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquid is 0.806g/L, namely 70.7%.

9. Detecting the purity of the freeze-dried powder sample by electrophoresis to 97.8 percent; the content of dopa in the freeze-dried powder sample is 0.1 percent by the content measurement of dopa;

The limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

In the comparative example, MMC composite cation exchange filler is not added in the enzymatic reaction process of the step 4, the impurities of a solution system are excessive in the enzymatic reaction process, and the enzymatic reaction efficiency is reduced, so that the dopa content of the final freeze-dried powder is less than 0.3 percent,

the corresponding color development results also show that the dopa content of the lyophilized powder sample of comparative example 1 is significantly lower than that of example 1 (fig. 1).

Comparative example 2

1. After fermentation of Pichia pastoris engineering bacteria is finished, placing the Pichia pastoris engineering bacteria in a tank, and placing the fermented liquid in a volume of 57.4L and passing through a high-speed centrifuge (room temperature)

Centrifuging at 4000rpm for 15min, discarding the precipitate, collecting supernatant with a volume of 36.5L, and collecting the supernatant to obtain crude feed liquid of target protein (recombinant type IV humanized collagen without dopa modification, i.e. intermediate).

3. And (3) ultrafiltering and concentrating the filtrate collected in the step (2) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), and collecting the trapped fluid when the conductivity of the trapped fluid is 0.44mS/cm by adding purified water, wherein the volume of the trapped fluid is 5L.

4. To the retentate collected in step 3, 5g of disodium hydrogen phosphate dodecahydrate, 1.65g of sodium dihydrogen phosphate dihydrate, 0.07g of copper sulfate and 0.02g of tyrosinase, designated 30U/mg, were added, stirred until completely dissolved, and then 1.3L of MMC complex cation exchange filler was added, pH was adjusted to 7.5 with sodium hydroxide, and stirred at 100rpm for 3.5 hours to perform an enzymatic reaction (room temperature).

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 3.8, and stirring for 1h (room temperature) at 100 rpm.

6. Filtering the feed liquid treated in the step 5 by using a 200-mesh screen, intercepting the MMC composite cation exchange filler on the screen to form a filter cake, leaching (room temperature) the filter cake by using 5L citric acid solution with pH of 3.7, and collecting the filler.

10mM, pH6.6 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer for 30min, filtering with 200 mesh sieve to collect eluate (i.e. filtrate), repeating the above soaking and filtering processes for 2 times with 1-time eluting filler trapped on the sieve, and combining the 3-time collected eluates to total 4.5L.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 91 mu S/cm, collecting 2.5L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 47.91g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquor is 0.835g/L, namely 73 percent.

9. Detecting by electrophoresis, wherein the purity of the freeze-dried powder sample is 92.3%; the content of dopa in the freeze-dried powder sample is 0.33 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

In the comparative example, the citric acid solution in step 6 was used in an amount of less than 5 times (6.5L) the filler volume, the eluted MMC composite cation exchange filler was insufficient, and impurities were not sufficiently removed, resulting in a final lyophilized powder purity of less than 95%.

Comparative example 3

1. After fermentation of Pichia pastoris engineering bacteria is finished, placing the Pichia pastoris engineering bacteria in a tank, and placing the fermented liquid in a volume of 57.1L and passing through a high-speed centrifuge (room temperature)

Centrifuging at 4000rpm for 15min, discarding the precipitate, collecting supernatant with a volume of 36.2L, and collecting the supernatant to obtain crude product feed liquid of target protein (recombinant type IV humanized collagen without dopa modification, i.e. intermediate).

3. And (3) ultrafiltering and concentrating the filtrate collected in the step (2) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), and collecting the trapped fluid when the conductivity of the trapped fluid is 0.38mS/cm by adding purified water, wherein the volume of the trapped fluid is 5L.

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 5.2, and stirring for 1h (room temperature) at 100 rpm.

10mM, pH6.6 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer for 30min, filtering with 200 mesh sieve to collect eluate (i.e. filtrate), repeating the above soaking and filtering processes for 2 times with 1-time eluting filler trapped on the sieve, and combining the 3-time collected eluates to total 4.4L.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 92 mu S/cm, collecting 2.4L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 37.68g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquor is 0.660g/L, namely 58%.

9. Through electrophoresis detection, the purity of the freeze-dried powder sample is 96.5%; the content of dopa in the freeze-dried powder sample is 0.32 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

In the comparative example, the pH of the feed liquid after the citric acid in the step 5 is adjusted is too high, and the target protein modified by the enzymatic reaction cannot be fully combined with MMC composite cation exchange filler, so that the amount of the finally obtained finished product protein is reduced, and the yield of fermentation liquor per liter is lower than 0.8g/L.

Comparative example 4

3. And (3) ultrafiltering and concentrating the filtrate collected in the step (2) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), and collecting the trapped fluid when the conductivity of the trapped fluid is 0.46mS/cm by adding purified water, wherein the volume of the trapped fluid is 5L.

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 2.9, and stirring for 1h (room temperature) at 100 rpm.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 92 mu S/cm, collecting 2.4L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 46.56g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquid is 0.815g/L, namely 72.1 percent.

9. Detecting by electrophoresis, wherein the purity of the freeze-dried powder sample is 93.7%; the content of dopa in the freeze-dried powder sample is 0.33 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

In the comparative example, the pH value of the feed liquid is too low after the citric acid in the step 5 is regulated, the target protein is degraded (dispersed degradation) after enzymatic reaction modification, and the purity of the finished product protein is reduced.

Comparative example 5

2. The supernatant collected in step 1 was clarified by filtration through a 0.45 μm hollow fiber filtration apparatus (room temperature), the retentate was diluted and filtered with 10L of purified water to recover the residual target protein, and the filtrate was collected to have a filtrate volume of 41.3L.

7. Immersing (at room temperature) the MMC composite cation exchange filler (i.e. the collected filler) treated in the step 6 in 4.5L

The eluate (i.e., the collected filtrate) was collected by filtration through a 200 mesh screen in 10mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH6.6 for 30 min.

8. And (3) ultrafiltering and concentrating the eluent collected in the step (7) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 91 mu S/cm, collecting 2.3L of the trapped fluid, loading the trapped fluid on a QFF chromatographic column (room temperature) well balanced by the purified water, collecting the column-penetrating liquid, and freeze-drying the obtained column-penetrating liquid to obtain 42.52g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquor is 0.746g/L, namely 67.1 percent.

9. Through electrophoresis detection, the purity of the freeze-dried powder sample is 97.2%; the content of dopa in the freeze-dried powder sample is 0.32 percent by the content measurement of dopa; the limit of endotoxin content of the freeze-dried powder sample is less than 1EU/mg by limulus reagent detection.

In the comparative example, the process of eluting the target protein modified by the enzymatic reaction in the step 6 adopts the buffer solution with the same total dosage to obtain the eluent by one-time immersion (i.e. elution), so that more residues are left on the surface of the filler, and the collection of the target protein eluent modified by the enzymatic reaction is insufficient, thereby reducing the amount of the final product protein and reducing the yield of fermentation liquor per liter to be lower than 0.8g/L.

Comparative example 6

1. After fermentation of Pichia pastoris engineering bacteria is finished, the Pichia pastoris engineering bacteria are placed in a tank, the volume of fermentation liquor is 57L, and the fermentation liquor is subjected to a high-speed centrifugal machine (room temperature)

Centrifuging at 4000rpm for 15min, discarding the precipitate, collecting supernatant with a volume of 36L, and collecting the supernatant to obtain crude product feed liquid of target protein (recombinant type IV humanized collagen without dopa modification, i.e. intermediate).

4. To the retentate collected in step 3 were added 4.6g of disodium hydrogen phosphate dodecahydrate, 1.63g of sodium dihydrogen phosphate dihydrate,

0.05g of copper sulfate and 0.02g of tyrosinase, designated 30U/mg, were stirred until completely dissolved, and conditioned with sodium hydroxide

The pH was brought to 7.5 and the enzymatic reaction was carried out with stirring at 100rpm for 3h (room temperature).

5. And (3) adding citric acid into the finished product stock solution obtained by the enzymatic reaction in the step (4) to adjust the pH to 3.5, and taking the mixture as chromatography liquid for standby.

6. Loading the feed liquid treated in the step 5 into an MMC composite cationic chromatographic column (room temperature) well balanced by 0.5% acetic acid solution, flushing impurities which are not combined with the filler by 0.5% acetic acid solution, flushing the chromatographic column (room temperature) by 10mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 6.5, and collecting target protein eluent subjected to enzymatic reaction modification for 3.4L in total.

7. And (3) ultrafiltering and concentrating the eluent collected in the step (6) by a 6KD roll-type filter membrane ultrafiltration system (room temperature), adding purified water until the conductivity of the trapped fluid is 91 mu S/cm, collecting 2.3L of the trapped fluid, and freeze-drying to obtain 46.26g of freeze-dried powder, wherein the yield of the recombinant IV-type humanized collagen finished product containing dopa per liter of fermentation liquor is 0.811g/L, namely the yield is 70.9%.

8. Detecting by electrophoresis, wherein the purity of the freeze-dried powder sample is 94.8%; the content of dopa in the freeze-dried powder sample is 0.12 percent by the content measurement of dopa.

The comparative example adopts the traditional process (i.e. the MMC column elution and separation of stock solution after enzymatic reaction), and the purity of the finished product protein is low and the dopa content is low.

In a word, the invention adopts biotechnology means to purify and modify the feed liquid expressed by the Pichia pastoris recombinant IV type collagen engineering bacteria fermentation, not only can obtain the recombinant IV type collagen with purity more than 95 percent, dopa content more than 0.3 percent, endotoxin content less than 1EU/mg and yield per liter fermentation liquor more than 0.8g/L, but also has simple purification process and is suitable for industrial production.

Claims

1. A purification production method of recombinant type IV collagen containing dopa is characterized by comprising the following steps: the method comprises the following steps:

after the fermentation of the pichia pastoris engineering bacteria for expressing the recombinant type IV collagen is finished, carrying out fermentation liquor pretreatment to obtain an intermediate material liquid containing the recombinant type IV collagen; mixing the intermediate product feed liquid with phosphate, copper sulfate, tyrosinase and MMC composite cation exchange filler, regulating pH to 7.4-7.8 by alkali, then carrying out enzymatic reaction by utilizing the tyrosinase in a liquid phase, regulating the raw liquid obtained by the enzymatic reaction to be acidic by using acid or acid solution after the enzymatic reaction is finished, enabling the recombinant IV-type collagen containing dopa formed after the enzymatic reaction modification to be replaced on the MMC composite cation exchange filler, eluting the MMC composite cation exchange filler by using acid solution to remove residual tyrosinase, eluting the recombinant IV-type collagen containing dopa replaced on the MMC composite cation exchange filler by using buffer solution, and carrying out aftertreatment on the obtained eluent to obtain the recombinant IV-type collagen containing dopa.

2. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: the pretreatment specifically comprises the following steps: and (3) after fermentation, placing the fermentation tank to obtain fermentation liquor containing recombinant IV-type collagen obtained by expression of pichia pastoris engineering bacteria, and sequentially carrying out solid-liquid separation, filtration clarification and ultrafiltration concentration on the fermentation liquor.

3. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: the recombinant IV type collagen expressed by the pichia pastoris engineering bacteria comprises one or more fusion sequence units composed of peptide fragments selected from natural IV type collagen.

4. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: in the pretreatment, an ultrafiltration concentration is performed by adopting a roll-type filter membrane ultrafiltration system with the thickness of less than or equal to 6KD, and the conductivity of trapped fluid is less than 0.5mS/cm.

5. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: the phosphate is disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and the addition amount of the copper sulfate, tyrosinase, MMC composite cation exchange filler, disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in the intermediate material liquid is 0.01-0.02 g/L, 100-200U/L, 25-30%, 0.9-1.1 g/L and 0.32-0.35 g/L in sequence; the specific conditions of the enzymatic reaction are as follows: stirring at 22-28 deg.c for 3-4 hr.

6. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: the replacement specifically comprises the following steps: and regulating the pH value of the stock solution obtained by the enzymatic reaction to 3.5-4.5 by using acid or acid solution, and stirring for 1-2 hours, wherein the acid is one of hydrochloric acid, sulfuric acid, citric acid and glacial acetic acid.

7. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: the acid solution adopted in leaching is acetic acid solution or citric acid solution with pH of 3.5-4; the specific conditions of the elution are as follows: the MMC composite cation exchange filler subjected to replacement and leaching is soaked in 10-15 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH value of 6.5-7 for more than one time, and the soaking time is more than or equal to 30min each time.

8. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 1, wherein: the post-treatment specifically comprises the following steps: and carrying out ultrafiltration concentration on the eluent, and then carrying out freeze drying, or carrying out ultrafiltration concentration on the eluent, and then carrying out endotoxin removal and freeze drying.

9. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 8, wherein: in the post-treatment, an ultrafiltration concentration adopts a roll-type filter membrane ultrafiltration system less than or equal to 6KD, the conductivity of trapped liquid is less than 0.1mS/cm, and an anion chromatographic column is adopted for endotoxin removal.

10. The method for purifying and producing recombinant type IV collagen containing dopa according to claim 8, wherein: the freeze-dried powder obtained by freeze-drying is recombinant type IV collagen with the purity of more than 95 percent and the content of the dopa is more than 0.3 percent.