ISOLATION AND PURIFICATION OF IMMUNE MODULATING LACTOFERRIN FROM MONGOL BOVINE COLOSTRUM
- VernacularTitle:ДАРХЛАА ДЭМЖИХ ҮЙЛДЭЛТЭЙ ЛАКТОФЕРРИН УУРГИЙГ ҮНЭЭНИЙ УУРАГТ СҮҮНЭЭС ГАРГАН АВАХ ТЕХНОЛОГИЙН СУДАЛГАА
- Author:
Chingunjav E
1
;
Jambal B
1
;
Amarsaikhan B
1
;
Gerelmaa T
1
;
Narantsetseg L
1
;
Sarantuya R
1
;
Bilegtsaikhan Ts
2
;
Purevjargal N
2
;
Tengis A
2
;
Javkhlan B
2
;
Tsendmaa Ts
2
;
Galindev B
2
;
Munkhtulga L
2
;
Nyambayar D
2
;
Munkhbat B
2
;
Baigalmaa B
2
Author Information
1. MNUMS School of Bio- Medicine and Pharmacy, Biochemistry and Laboratory
2. MNUMS Core Laboratory, Science Technology Center
- Publication Type:Journal Article
- Keywords:
Lactoferrin, Mongol bovine colostrum, HPLC, ion exchange chromatography
- From:Innovation
2017;11(1):30-33
- CountryMongolia
- Language:Mongolian
-
Abstract:
BACKGROUND
Bovine colostrums is the milk secreted by cows during the first few days after parturition. It
contains many essential nutrients and bioactive components, including growth factors,
immunoglobulins, lactoperoxidase, lactoferrin and cytokines ets. Lactoferrin has been reported
for its multifunctional properties such as antifungal, antibacterial, antiviral antioxidant and
anticancer activities. The aims of this study focused on the isolation and purification of lactoferrin
from Mongolian bovine colostrums. Lactoferrin purified using HiTrap DEAE an ion exchange
chromatography. Lactoferrin purification efficiency was about 60.5%. The single band of purified
lactoferrin has been observed in SDS-PAGE electrophoresis.
METHODS
Bovine colostrum was collected at a cow farm in the Darkhan province of Mongolia. At first
the cream was separated by centrifugation (10000 xg 20 min at 4oC). In order to separate the
whey, the samples were precipitated with 1mol/l to pH 4.6 and centrifuged at 10000 g 20 min
again. The samples of whey were stored at -18oC to the analysis. Lactoferrin was purified by
HiTrap DEAE an ion exchange chromatography using 0.005 M phosphate buffer (pH 7.7) and
linear gradient NaCl from 0.25M, 0.5M, 1M. During chromatography, protein in the eluents was
monitored by ultraviolet absorbation at 280 nm with the instrument. Purity test done by using
polyacrylamide gel electrophoresis under denaturated condition (SDS-PAGE) method by Laemmli
(1970). For HPLC determination of the lactoferrin by Shimadzu Nexera X2 HPLC system with UV/
VIS detector were used. Detection was carried out at the wavelength 280 nm. Separation was
performed on a chromatographic column Protein R C18 ,2.2 x 150 mm, 5 μm particle size. Linear
gradient and flow rate 0.2 ml/min were used. Mobile phase a consisted of water / acetonitrile/
trifluoroacetic acid ( 95:5:0.1). The column temperature was set at 40oC and injection volume
was 10 μl. Data were collected and evaluated by software Lab Solution. An external standard
method for quantification analytes was used.
RESULTS
Purified lactoferrin in the present study had a good concentration and purification efficiency
was about 60.5 %. Protein fraction from 1M NaCl gradient delivers sharp and clean peak to
HPLC chromatogram that fits intensity and retention time of standard bovine lactoferrin.
Ammount of lactoferrin in bovine colostrums was 0.6 mg/ml and it`s molecular weight 80 kDa as
a standard sample. The retention time of lactoferrin fraction which is purified by SDS-PAGE gel
electrophoresis. The peak of fraction same compared to the standard lactoferrin 5.8 minutes
by HPLC analysis.
CONCLUSION
Ion exchange chromatography shows reliable and easy isolation of lactoferrin from Mongol
bovine colostrum.
