Research Paper

Volume : 4 | Issue : 6 | June 2015 ISSN No 2277 - 8179

Biology

A Comparison of Protein Extraction

KEYWORDS : Aspergillus fumigatus, Pro-

tein precipitation, Secretome, Secretory

Sanyukta

Department of Biotechnology, St. Xaviers College, Ranchi.

Srivastava A K

Department of Botany, St. Xaviers College, Ranchi.

ABSTRACT

Sample preparation is a fundamental step in proteomic methodologies. Protein precipitation is frequently used to
concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied,
which rely on different principles, have been compared for precipitation of extracellular proteins of Aspergillus fumigatus strain (MTCC 1811)
as Aspergillus species comprise strains of medical and industrial importance. Secretory proteins were extracted using acetone, 2-propanol
and chloroform/methanol when grown under different carbon sources. It was found that chloroform/methanol delivered the highest protein
recovery of 86.780.57%, 85.550.59%, and 85.271.07% when lactose, glucose and sucrose respectively were used as sole carbon source. The
lowest percentage recovery of extracellular proteins was 67.681.44%, when proteins were extracted using acetone from the filtrate of culture
grown under sucrose as sole carbon source.

Introduction:
Filamentous fungi have received attention for protein production because of their high secretion capability and eukaryotic
posttranslational modifications. Aspergillus species such as A.
niger and A. oryzae are known for their exceptional ability to secrete large amounts of homologous enzymes. For decades they
have been commonly exploited as commercial production organisms for a variety of enzymes [1, 2]. Sample preparation is
crucial for conducting reliable proteomic analysis [3, 4]. Samples should have a high-protein concentration and be free of salt
and other interfering components, such as detergents, nucleic
acids, lipids, etc [5, 6]. Precipitation is widely used for processing of biological molecules such as proteins to concentrate and
fractionate the target molecule from various contaminants [7].
For example, in the biotechnology industry, protein precipitation is used to eliminate contaminants commonly contained in
blood [8]. The first step is usually homogenisation or sonication
followed by protein precipitation and solubilisation in a suitable
buffer. Chloroform, methanol, acetone and isopropanol are common organic solvents used as protein precipitating reagents [9].
Fungi secrete extracellular proteins or enzymes to enable them
to harvest nutrients from the environment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. The
knowledge of secretome not only opens path for study of pathogenesis but also for diagnosis [10, 11, 12]. The secretion pattern
in different conditions will give an insight for efficient production of specific secreted proteins [13]. It will lighten the different prospects in industrial production of such proteins. In this
study, we investigated the efficiency of various methods for extracellular protein precipitation of Aspergillus fumigatus strain
(MTCC 1811). Proteins were extracted using acetone, 2-propanol and chloroform/methanol when grown under different
carbon sources. Some proteins were lost during sample preparation; precipitation followed by re-solubilisation in sample solution rarely gives a 100% yield.
MATERIALS & METHODS
Fungal culture
The fungal strain Aspergillus fumigatus strain (MTCC 1811) was
obtained from Microbial Type Culture Collection, Institute of
Microbial Technology, Chandigarh. The culture was grown in
Czapek dox medium under three different carbon sources, glucose, lactose and sucrose. Each experiment was performed in
triplicate. 100 ml Flasks containing 50 ml Czapek dox medium
for each sugar were inoculated with 106 spores ml1 suspension.
The flasks were incubated at 30C in a rotary shaker at 125rpm
for five days. Culture was then centrifuged at 5,000 g for 15
min, and the supernatant was filtered through a dried Whatman
708

IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH

(No.1) filter paper and filtrate was used for protein analysis.
Protein precipitation
Three different precipitation procedures were used: acetone, 2propanol and a mixture of chloroform and methanol.
Chloroform/methanol precipitation
Experiments were performed at room temperature. Four volumes of methanol were added to one volume of the protein
sample, and the mixture was vortexed. One volume of chloroform was then added, and the mixture was vortexed. The sample
was centrifuged at 10000g for 5min and the aqueous methanol
layer was removed from the top of the sample. The proteins remained at the phase boundary between the aqueous methanol
layer and the chloroform layer. Four volumes of methanol were
added, and the mixture was vortexed. The sample was spun at
10000g for 15min. The supernatant was removed without disturbing the pellet, and the pellet was air dried.
Acetone precipitation
Experiments were performed at 4C. Four volumes of ice-cold
acetone containing 20mM DTT were added to one volume of
protein sample. The mixture was vortexed and incubated at
20C for 1h. This was followed by centrifugation at 10000g for
15min at 4C. The supernatant was discarded and the pellet was
air dried.
2- propanol precipitation
Equal volume of filtrate was combined with 100% isopropanol.
After vigorous mixing (1min) the samples were incubated overnight at 4oC. Then it was centrifuged at 10000g for 15 min. The
supernatant was removed carefully without disturbing the pellet,
and the pellet was air dried.
Determination of protein concentration
The protein concentration of the re-solubilised samples was determined in triplicate using the Lowry assay [14]. The efficiency
of precipitation was determined as a ratio of the protein concentration before and after precipitation. The results presented are
an average of three experiments.
Results:
In order to obtain reliable, reproducible and significant data
proper sample preparation is crucial, particularly in comparative
proteomic studies where minor differences between experimental and control groups are often meaningful [15]. In this study,
we compared different carbon sources on protein secretion.
These methodologies are useful for identifying changes in extracellular protein expression under different experimental condi-

Research Paper

Volume : 4 | Issue : 6 | June 2015 ISSN No 2277 - 8179

tions. It was found that chloroform/methanol delivered the highest protein recovery of 85.550.59%, 2-propanol 79.500.35% and
acetone 67.840.49% when glucose was used as carbon source
[Table1].

Figure 1 Comparison of the precipitation efficiency of different methods for various carbon sources.

Table1 Percentage of secretory protein recovery after various precipitation procedures when glucose was used as sole
carbon source.
Precipitation Protein
amount before
method
precipitation(g)
Acetone
144.541.69
2- propanol 144.541.69
Chloroform/ 144.541.69
methanol

Protein
amount after
precipitation(g)
98.061.20
114.91.71

Percentage of
recovery
(%)
67.840.49
79.50.35

123.661.49

85.550.59

Values are the meanstandard deviation of three independent

154.942.55

107.162.25

69.150.51

2- propanol

154.942.55

125.12.12

80.730.135

Chloroform/ 154.942.55
methanol

134.61.82

86.780.57

Values are the meanstandard deviation of three independent

144.782.77

98.061.20

67.681.44

2- propanol

144.782.77

114.261.59

78.920.60

Chloroform/ 144.782.77
methanol

123.431.27

85.271.07

Values are the meanstandard deviation of three independent

When lactose was used as sole carbon source percentage recovery was of 86.780.57% for chloroform/methanol, for 2-propanol
it was found to be 80.730.13% and acetone showed 69.150.51%
recovery [Table2]. Percentage recovery of extracellular proteins
was 85.271.07% for chloroform/methanol, for 2-propanol it was
found to be 78.920.60% while acetone showed 67.681.44% [Table 3]. The trend of precipitation efficiency was similar in the
three different culture conditions [Figure 1].
Discussion
The comparison of protein content in three filtrate revealed that
the acetone method produced the lowest efficiency for protein
precipitation. Reduced protein loss ( fewer proteins in supernatants) occurred with the 2-propanol and methanol/chloroform
methods. The methanol/chloroform method yielded the lowest
loss of proteins, as determined by analysis of the medium filtrate. The acetone method yielded low precipitation efficiency
in comparison to precipitation with methanol/chloroform. The
study by Ewelina Fic et al also showed that the precipitation
with chloroform/methanol delivered the highest protein recovery for proteomic analysis [16]. However, as has been shown by
Garcia-Rodriguez S et al [6] acetone precipitation following incubation with DTT gave the highest protein recovery. In summary, it is important that the chosen protein precipitation method
is able to effectively concentrate samples and eliminate contaminants; however, precipitation procedures rarely yield complete
recovery.

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