Recombinant Protein Expression is a very important technology for the production of Biopharmaceuticals. The expression of recombinant proteins in different host systems has always been a major feature and challenge in the Biotechnology industry (Baldi, L et al 2006). The most important criteria for selecting the host system is that the cell line must have the stability of expression. There are several expression systems but the majority of the recombinant proteins are produced in either Mammalian cells or in E.Coli.
Mammalian cells are the most common preferred hosts for recombinant protein production because of their stable gene expression, correct protein folding, high levels of protein expression, glycosylation patterns and post translational modifications (Wurm, F.M. 2009). There are various mammalian expression systems namely Chinese hamster ovary (CHO) cells, Nonsecreting (NSO) myeloma cells, Baby hamster kidney cells (BHK) and PER.C6 Human embryonic retinal cell line (Dickson, A.J. et al, 2002). The Chinese hamster ovary are the most important cell lines for the production of recombinant proteins (Hacker, L.D 2009). .
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Glucose is utilised by the cells for energy. The majority of the glucose in the body enters the normal Glycolysis pathway (Fig 1) whereas the unused glucose enters the Polyol Pathway (Fig 1). The Polyol pathway also called as the sorbitol or Aldose Reductase pathway is thus one minor pathway of glucose metabolism. The polyol pathway is a two step enzymatic pathway. In the first step glucose is first reduced to sorbitol using nicotinamide adenine dinucleotide phosphate (NADPH). This conversion of glucose to sorbitol is the rate determining step of the pathway (Kinoshita and Nishimura, 1988). Aldose Reductase (AR) is the first and rate limiting enzyme in this pathway.
Further in the second step sorbitol is oxidised to fructose using nicotinamide adenine dinucleotide (NAD+) and the enzyme facilitating this conversion is sorbitol dehydrogenase (SDH).
Sorbitol which is an alcohol that is strongly hydrophilic diffuses through the cell membrane at a much slower rate than glucose and fructose. As a result it tends to accumulate intracellularly and thus causes an increase in the osmotic pressure. The increase in osmotic pressure causes the cells to undergo apoptosis. The polyol pathway tends to become active when the intracellular glucose levels are being elevated. When the glucose levels in the blood are normal this conversion will not cause any problem as AR has low affinity for glucose at normal concentrations. However in hyperglycemic state there is a rise in the affinity levels of AR for glucose. This rise leads to the accumulation of sorbitol and eventually leads to the activation of the Polyol pathway.
Figure 1: Glycolysis and Polyol Pathway
The polyol pathway appears to be implicated in diabetic complications that lead to the damage of the nervous tissue and also to the retina and the kidney. Thus if the polyol pathway is inhibited then there will be no conversion of the unused glucose. Thus there will be an increase in the amount of glucose entering the glycolysis pathway and eventually more amount of energy released. Inhibiting the first step of the polyol pathway is also important as the conversion of glucose to sorbitol can be stopped.
In order to completely understand the first step of the polyol pathway I have studied the expression and detection of Aldose reductase in a new mammalian cell line.
2. Materials and Methods:
2.1 Cell line and Growth medium:
In this study a new cell line namely LB01 from Lonza Biologics has been used. The cell line was grown in CD CHO (Invitrogen) medium. The CD CHO is a chemically defined protein free medium. The cell lines were grown as suspension cultures in this medium. Methionine Sulfoximine (MSX) was added to the medium to culture the cell lines. 25µM of MSX was added in order to maintain a selective pressure. LB01 cells at 0.2 - 106 cell/ml were cultured in a 50ml PETG Sterile flask and maintained in an Unitron shaking incubator at 37°C and 100rpm.
2.2 Determination of Cell viability:
The cell number and viability were measured by Trypan blue exclusion. 20µl of 1% (w/v) trypan blue was mixed with 20µl of the cell suspension and then loaded onto the haemocytometer. The number of live and dead cells were counted by using light microscopy so that the total and the viable cell numbers could be compared.
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2.3 Growth Analysis and Subculturing:
LB01 cells were grown as suspension cultures and the cell density was measured for upto 14 days in order to analyse the growth pattern of the cells. The cells were routinely subcultured during the mid-exponential phase of batch culture that is usually every three to four days.
2.4 Protein Extraction for Enzymatic Assay:
5ml of the cells during the day 5 of their growth phase were taken in a 10ml BD Falcon tube. The tubes were now centrifuged at 10,000 - g for 20 min at 4°C. The resulting pellet was washed twice with 3ml of PBS. The pellet was again centrifuged so as to remove the remaining PBS. 200µl of the extraction buffer was then added to the pellet. The extraction buffer consisted of 25Mm Tris (pH 7.4), 50mM Nacl, Aprotinin (10µg/ml), Leupeptin (10µg/ml) and PMSF (0.57mM). The pellet along with the extraction buffer was vortexed. The sample was then freeze-thawed in Liquid Nitrogen for 4 times and then centrifuged at 10,000 - g for 20 min at 4°C. The supernatant was collected and stored in 20µl aliquots at -80°C.
2.5 Enzymatic Assay:
For the enzymatic assay , each 1ml cuvette contained 0.15M Potassium Phosphate buffer (0.33ml), 0.7M Glucose 9 (0.1ml), 10mM NADPH (10µl), cell extract, and water (0.55ml). The absorbance was measured in a spectrophotometer at 340nm. The decrease in absorbtion was measured every 30 seconds for the first 10 min and this decrease was taken as the measure of NADPH oxidized.
2.6 Protein extraction for Western Blot:
2ml of the cells at a concentration of 5 - 106 cells/ml are taken in falcon tube and centrifuged at 10,000 - g for 20 min at 4°C. The resulting pellet was washed with PBS twice and recentrifuged to remove any PBS remaining. The pellet is now resuspended in RIPA buffer (300µl), Aprotinin (3µl), Leupeptin (3µl) and PMSF (3.5µl). The sample mixture was then incubated on ice for 30 min. Following incubation the sample is centrifuged at 10,000 - g for 20 min at 4°C. The supernatant is collected and stored as 25µl aliquots at -80°C.
2.7 Biorad Protein Assay:
The biorad assay for determining the protein concentration was performed in a 96 well plate. The assay was performed according to the normal laboratory protocol and the absorbance was measured on a plate reader at 570nm.
2.8 Western Blot:
The western blot technique was carried out as per the normal protocol. A 12.5% Separating and stacking gel was used once the gel is completely set it is placed on the running stand. The protein samples were prepared accordingly with the sample buffer and 30µl of the protein sample was loaded onto the gel. The protein samples were loaded onto the gel in order along with the standard protein marker. The gel was run at 60v until the dye front has just crossed the stacking layer and then the current was increased to 200v and run for 40 min. Once the gel has run completely the gel is now transferred onto the nitrocellulose membrane on a trans-blot apparatus. The gel was transferred at 15v for 30 min. The membrane was further stained with Ponsceau stain and then blocked with 3% Milk solution for a minimum of 1hour. The membrane is later incubated with the primary antibody and then secondary antibody. The Aldose Reductase (FL-316) from Santa Cruz Biotechnology was used
as the primary antibody and was diluted at a range of 1:1000. Anti-rabbit IgG HRP was used as a secondary antibody. The JAR cell lysate from Santa Cruz Biotechnology was used as a positive control for the detection of the protein. The membranes were at last subjected to Enhanced Chemiluminescence (ECL) for 2 mins and for 5 mins respectively.
3.1 Growth Curve of LB01 cells:
LBO1 cells were batch cultured as suspension cultures and the growth rate of the LB01 cell line had been measured for a period of 14 days. Each day the cells were counted by trypan blue exclusion using a light microscope from which the viable cell density was calculated. It has been observed that the cells grow at an exponential phase till they reach their maximum viable cell density on Day 7 after which they enter the decline phase. The viable cell density decreases gradually after day 7 and at a certain point of time (Day 13) it eventually becomes zero. It is thus highly recommended that the subculturing of the cells is done on day 4 or day 5 as the cells are in the mid-exponential phase of batch culture. The values thus obtained have been represented graphically (Fig. 2).
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Figure 2: Growth curve of LB01 cell line. The viable cell density of the cell line was calculated by trypan blue exclusion.
Figure 3: Percentage of Viable cells
3.2 Enzymatic Assay Analysis:
The enzymatic assay was set up as described previously in section 2.4. The change in NADPH value was spectrophotometrically monitored in the presence of glucose as well as in the absence of glucose. In order to analyse the rate of decrease of NADPH three concentrations of the cell extract (5µl, 10µl and 20µl) were added to the assay. The rate of decrease in the values of NADPH in the presence of glucose is more when compared to the values without glucose. This decrease in values clearly states that NADPH reduces glucose to sorbitol in the polyol pathway.
Table 1 (A): Enzyme activity values with 5µl of the extract in the presence and in the absence of Glucose
In the presence of Glucose
In the absence of glucose
Table 1. (B): Enzyme activity values with 10µl of the extract in the presence and absence of Glucose
In the presence of Glucose
In the absence of glucose
Table 1 (C): Enzyme activity values with 20µl of the extract in the presence and absence of Glucose
In the presence of Glucose
In the absence of glucose
3.4 Western Blot:
To examine the expression of Aldose Reductase Western Blot analysis was carried out with the enzyme extracted as described in section 2.6. The Biorad Protein assay was first performed and the amount of protein sample to be loaded onto the gel was determined. The protein concentration was 8.12µg/µl in LB01 cell extract. Aldose Reductase (FL-316) was used as the primary antibody for detecting Aldose Reductase protein in LB01 cells. Anti rabbit IgG HRP was used as the secondary antibody and after Enhanced Chemiluminescence (ECL) a band of 37kDa which cconfirms the presence of Aldose Reductase was obtained. The membrane was subjected to 2mins and 5 mins exposure to ECL. The bands obtained (Fig. 4(a) and 4(b)) clearly indicate that there is a slight darker band observed in the film that is exposed to ECL for 5 mins.
1 2 3 4 5 6
Figure 4 (a): 2 mins exposure to ECL.
1 2 3 4 5 6
Figure 4 (b): 5 mins exposure to ECL.
Lanes 1: Protein Marker, Lane 2: JAR cell lysate (Positive control), Lane 3: Cell line A, Lane 4: Cell line B, Lane 5: Cell line C, Lane 6: LB01 Cell line
Aldose Reductase is a member of the aldo-keto reductase family. The aldose reductase has broad substrate specificity. The experiments that have been done clearly demonstrate that there is a positive correlation between NADPH and Aldose Reductase. It is thus confirmed that that glucose is reduced to sorbitol with the oxidation of NADPH TO NADP+. Consequently though there is a decrease in absorbance of NADPH values in the enzymatic assay the rate of decrease is not constant. The reason may be that the enzyme present in the cells may be not sufficient for the assay. Previous studies of Aldose Reductase experiments showed a gradual rate of decrease in NADPH absorbance when performed with Rats lens (Saraswat, M. Et al, 2008). Other studies on ischemic rat heats (Hwang, C. Y., et al. 2001) have also resulted in positive results for the aldose reductase assay. These studies give us an idea that there is probably not enough amount of enzyme present in the enzymatic extract.
In order to move forward and firmly confirm the presence of Aldose Reductase in LB01 cells the enzyme extracts for western blotting have been prepared. The positive results from western blot analysis that is the appearance of the band at 37kDa confirms the presence of Aldose Reductase. JAR Cell lysate (positive control) which was also loaded onto the gel also gives us an affirmation of the Aldose reductase protein.