RU2763552C1 - Method for isolation of camel lactoferrin - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. A method is proposed for the isolation of camel lactoferrin, including the centrifugation of camel milk and the removal of the formed fat fraction. Next, heat treatment of the fat-free fraction of camel milk is carried out, the resulting fraction is subjected to centrifugation, and the supernatant is subjected to affinity chromatography on immobilized lectin of the river waterworm, for which the entire volume is passed through a column of lectin-sepharose, equilibrated with tris-HCl (pH=8.0) with a buffer, fraction obtained at the previous stage. Then the column is washed with 1 M sodium chloride solution buffered with tris-HCl buffer (pH=8.0), and then the camel lactoferrin bound on the column is eluted with 0.1 M lactose solution in borate buffer pH = 9.0. The resulting eluate is dialyzed and concentrated.

EFFECT: method allows to obtain electrophoretically and chromatographically homogeneous preparation of camel lactoferrin.

1 cl, 2 dwg, 1 tbl

Description

The invention relates to the field of medicine, namely biochemistry, and can be used, in particular, for the isolation of camel lactoferrin.

In practice, a method is known for obtaining a purified preparation of camel lactoferrin, based on a combination of high performance liquid chromatography and tandem mass spectroscopy.(Xia Li , Zengmei Li , Enmin Xu , Ling Chen , Hua Feng , Lu Chen 1, Ligang Deng, Dongliang Guo. Determination of Lactoferrin in Camel Milk by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry Using an Isotope-Labeled Winged Peptide as Internal Standard. Molecules 2019-v.24, p.4199=4208; doi: 10.3390/molecules24224199) or a known method (prototype) based on a combination of centrifugation methods and various types of chromatography (Redwan EM, El-Fakharany EM, Uversky VN, Linjawi MH Screening the anti infectivity potentials of native N- and C-lobes derived from the camel lactoferrin again hepatitis C virus BMC Complement Altern Med 2014 Jul 3;14:219 doi: 10.1186/1472-6882-14-219.).

The disadvantages of the known methods are:

- low product purity

-low efficiency and low product yield;

- the duration of the event;

- use of expensive equipment

The invention is aimed at increasing the degree of purification, accelerating and reducing the cost of the method for purification of camel lactoferrin.

This technical result is achieved by heat treatment of the fat-free fraction of camel milk at 75-77°C for 30 minutes, the resulting fraction is subjected to centrifugation, and the supernatant is subjected to affinity chromatography on immobilized lectin of the river bodyagi, for which purpose through a column of lectin-Sepharose, balanced Tris-HCl (pH=8.0) buffer, pass the entire volume of the fraction obtained in the previous stage, then the column is washed with 1M sodium chloride solution buffered with Tris-HCl buffer (pH=8.0), and then elute bound on camel lactoferrin column with 0.1 M lactose solution in borate buffer pH=9.0, the resulting eluate is dialyzed and concentrated.

Purification of lactoferrin was carried out according to the proposed method. We used milk of camels with the optimal concentration of lactoferrin (7-28 days of lactation). The work used 93 samples of camel milk, which were obtained from camel farms in the Krasnoyarsk and Kharabalinsky districts of the Astrakhan region in October-November 2018, with a total volume of 550.0 ml. At the first stage, milk samples were centrifuged at 8000 rpm for 40-45 minutes and then the resulting fat fraction was removed. The fat-free fraction was labeled, the concentration of lactoferrin was determined and stored at -18°C. At the next stage, 450 ml. fat-free fraction of camels' milk was subjected to heat treatment of the entire used volume of the fat-free fraction of camels' milk at 75-77°C for 30 minutes in a temperature-controlled cell of an AM-208 autothermostat. Further, the resulting fraction was subjected to centrifugation at 8000 rpm for 40 min in a CLS-32 centrifuge. The supernatant was collected and used in further work. The final step of CLF purification is affinity chromatography of camel lactoferrin on immobilized lectin of the river bog. The entire volume of the fraction obtained at the previous stage was passed through a column (1.5x12 cm) of lectin-sepharose, balanced with Tris-HCl (pH=8.0) buffer. Next, the column was washed with 1M sodium chloride solution buffered with Tris-HCl buffer (pH=8.0), and then eluted bound on the CLF column with 0.1 M lactose solution in borate buffer pH=9.0. The resulting eluate was dialyzed and concentrated to a volume of 26.0 ml.

Below, table 1 provides information on a comparative study of the selection of camels lactoferrin by the method of the prototype (method 1) and affinity chromatography with lectin bodyagi river (method 2). The analysis of the obtained results showed that affinity chromatography on the lectin of the river bodya not only significantly simplified and reduced the time for isolation and purification of camelid lactoferrin, but also significantly increased the yield of the target product from 27.31% to 69.3%. The degree of purity has also increased.

drug from 91.1% to 98.3%. Objectively, these figures confirm

electrophoresis in polyacrylamide gel (figure 1). The preparation shows the presence of protein impurities in lactoferrin obtained by the method of the prototype.

The degree of purification of camel lactoferrin was 36.8, and the yield was about 70%. In total, 321.0 mg of pure lactoferrin was obtained from 550 ml of camel milk, which was stored at -24°C until further use.

The essence is illustrated by drawings, where:

Fig.1. Electrophoresis in polyacrylamide gel. 1 - fat-free fraction of camels milk; 2 - lactoferrin camel obtained by the method of the prototype; 3 - camel lactoferrin obtained by the proposed method.

Fig.2. Reversed phase high performance liquid chromatography of a purified preparation of camelid lactoferrin. Silosorb S-8 column 115.0x3.5 mm Elution with 0.1% TFA and acetonitrile gradient from 0 to 60%.

Below are the results of the study.

Example #1

At the first stage, milk samples with a volume of 500.0 ml were centrifuged at 8000 rpm for 45 minutes and then the resulting fat (oil) fraction was removed. The fat-free fraction was labeled, the concentration of lactoferrin was determined and stored at -18°C. At the next stage, 450 ml. fat-free fraction of camels' milk was subjected to heat treatment of the entire used volume of the fat-free fraction of camels' milk at 77°C for 30 minutes in a temperature-controlled cell of an AM-208 autothermostat. Further, the resulting fraction was subjected to centrifugation at 8000 rpm for 40 min in a CLS-32 centrifuge. The supernatant was collected and used in further work. The final step of CLF purification is affinity chromatography of camel lactoferrin on immobilized lectin of the river bog. The entire volume of the fraction obtained at the previous stage was passed through a column (1.5x12 cm) of lectin-Sepharose equilibrated with Tris-HCl (pH=8.0) buffer. Next, the column was washed with 1 M sodium chloride solution buffered with Tris-HCl buffer (pH=8.0), and then eluted bound on the CLF column with 0.1 M lactose solution in borate buffer pH=9.0. The resulting eluate was dialyzed and concentrated to a volume of 22.0 ml.

Example #2

At the first stage, milk samples with a volume of 400.0 ml were centrifuged at 8000 rpm for 40 minutes and then the resulting fat (oil) fraction was removed. The fat-free fraction was labeled, the concentration of lactoferrin was determined and stored at -18°C. At the next stage, 360 ml. fat-free fraction of camels milk was subjected to heat treatment of the entire used volume of fat-free fraction of camels milk at 75°C for 30 minutes in a thermostated cell of an AM-208 autothermostat. Further, the resulting fraction was subjected to centrifugation at 8000 rpm for 40 min in a CLS-32 centrifuge. The supernatant was collected and used in further work. The final step of CLF purification is affinity chromatography of camel lactoferrin on immobilized lectin of the river bog. The entire volume of the fraction obtained at the previous stage was passed through a column (1.5x12 cm) of lectin-Sepharose equilibrated with Tris-HCl (pH=8.0) buffer. Next, the column was washed with 1 M sodium chloride solution buffered with Tris-HCl buffer (pH=8.0), and then eluted bound on the CLF column with 0.1 M lactose solution in borate buffer pH=9.0. The resulting eluate was dialyzed and concentrated to a volume of 20.0 ml.

Suggested way:

- allows to obtain electrophoretically and chromatographically homogeneous preparation of camel lactoferrin;

-increases efficiency (increases the yield of the final product by 42%),

- shortens the release time of camel lactoferrin

-Easier cleaning process

Claims (1)

A method for isolating camel lactoferrin, which includes centrifuging camel milk at 8000 rpm for 40-45 minutes and then removing the resulting fat fraction, characterized in that further heat treatment of the fat-free camel milk fraction is carried out at 75-77 ° C for 30 minutes, the resulting fraction is subjected to centrifugation, and the supernatant liquid is subjected to affinity chromatography on immobilized lectin of the river bodyagi, for which purpose the entire volume of the fraction obtained at the previous stage is passed through a lectin-sepharose column equilibrated with Tris-HCl (pH = 8.0) buffer, then the column washed with 1 M sodium chloride solution buffered with Tris-HCl buffer (pH=8.0), and then the camel lactoferrin bound on the column is eluted with 0.1 M lactose solution in borate buffer pH=9.0, the resulting eluate is dialyzed and concentrated.

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