Isolation and characterization of serum albumin from Camelus dromedarius

Abstract

Serum albumin constitutes 35-50 mg/ml of plasma proteins and performs various physiological activities including the regulation of osmotic pressure on blood, maintaining buffering of the blood pH, carrying different fatty acids and other small molecules, such as bilirubin, hormones, drugs and metal ions, as well as participating in immunological responses. Serum albumin is an extensively used protein in biotechnological and pharmaceutical industries. The camel (Camelus dromedarius) is well tailored to successfully survive in extremely hot and dry climates. Plasma osmolality in the camel increases during water-deprived conditions. In such circumstances serum albumin is crucial in the regulation of blood pressure. The study of biochemical, biophysical and immunological aspects of camel serum albumin (CSA) are likely to provide molecular insights into camel physiology and may render it an alternative to human serum albumin (HSA) and bovine serum albumin (BSA) in all cases. However, these proteins are currently not available or cannot be utilized due to a variety of considerations. In this study, 12 mg of highly pure CSA was obtained from 1 ml plasma. Coomassie Brilliant Blue staining of SDS-PAGE yielded one band and RP-HPLC results revealed a single sharp peak, indicating homogenous preparation of the CSA. The charge/mass ratio and surface hydrophobicity of the CSA was similar to that of BSA. Mass spectrometry analysis of the purified protein confirmed the identity of CSA.

Keywords: camel serum albumin; capillary electrophoresis; high-performance liquid chromatography; protein purification and characterization.

Figure 1.

Capture of camel serum albumin (CSA) by affinity chromatography. (A) Elution profile of CSA from Cibacron Blue 3G (Blue-Sepharose). Plasma diluted 10-fold was loaded on Blue-Sepharose. The CSA bound matrix was washed with 20 mM Tris-HCl, pH 8.0. Bound protein was eluted with NaCl linear gradient (dotted line). The eluted protein is shown by the solid line. Following analysis of the purity of different eluted fractions on SDS-PAGE, peak 1 and shoulder peak 2 were pooled separately. Fractions in peak 1 were more pure than fractions in peak 2. (B) Lane 1, low molecular weight (LMW) markers; lane 2, pooled peak 1 from Blue-Sepharose was loaded on 12% SDS-PAGE.

Figure 2.

Purification of camel serum albumin (CSA) by Q-Sepharose. (A) Peak 1 eluted from Blue-Sepharose was pooled and extensively dialysed against 20 mM Tris-HCl, pH 8.0 before binding on Q-Sepharose. Washing was carried out using 20 mM Tris-HCl, pH 8.0. Bound protein was eluted with NaCl linear gradient (dotted line). Eluted protein is shown by the solid line. Following analysis of the different fractions on 12% SDS-PAGE, relatively pure fractions (present in the 3rd peak) were pooled for further purification. (B) SDS-PAGE analysis. Lane 1, low molecular weight (LMW) markers; lane 2, pooled peak 3 eluted from Q-Sepharose was loaded on 12% SDS-PAGE.

Figure 3.

Polishing of camel serum albumin (CSA) by gel filtration chromatography. (A) Pooled 3rd peak from Q-Sepharose was loaded on Sephacryl S-100. The solid line is the eluted protein from Sephacryl S-100. Eluted fractions were analyzed on 12% SDS-PAGE for purity. (B) SDS-PAGE analysis of purified CSA. Lane 1, low molecular weight (LMW) markers; lane 2, pooled peak 2 from Sephacryl S-100.

Figure 4.

Multiple sequence alignment. Partial sequence of camel serum albumin (CSA) (NCBI accession no. HM640019.1) was aligned with human (P02768), bovine (P02769), horse (P35747) and rabbit albumin (P49065) using MAFFT multiple sequence alignment program. Residues are color coded according to conservancy. Boxed albumin sequences correspond to peptides identified by mass-spectrometry.

Figure 5.

High-performance liquid chromatography (HPLC) analysis of camel serum albumin (CSA) and bovine serum albumin (BSA). Homogeneity of the purified CSA and surface hydrophobicity of the CSA was compared with commercial BSA by HPLC analysis. (A) Purified CSA and (B) commercial BSA were loaded on C18 column. Bound CSA was eluted in the single peak and corresponded to the same retention time as BSA, indicating similar surface hydrophobicity in the two albumins.

Figure 6.

Capillary zone electrophoresis analysis of camel serum albumin (CSA) and bovine serum albumin (BSA). Charge to mass ratio of CSA was compared with BSA in capillary zone electrophoresis mode. (A) Purified CSA and (B) commercial BSA was passed through polyvinyl alcohol (PVA)-coated capillary at pH 3.6. Purified CSA was eluted in a single symmetrical peak and the retention time of CSA and BSA were similar, indicating a similar charge/mass ratio.