Biotechnological
Communication
Biosci. Biotech. Res. Comm. 9(2):
Purification of recombinant subunit vaccine by HbsAg cell lysis using various enrichments of chaotropic and
A. Ali, Z H Khan, M K Sahib, S Durrani, U Shaligram, EB Raju and A. Durrani
Department of Biochemistry, Shri Shivaji College, Akola, India
ABSTRACT
Hansunella polymorpha is extensively used as production strain for expressing recombinant proteins including recom- binant Hepatitis B surface antigens (HBsAg). HBsAg is expressed in H. polymorpha and remains bounded to the mem- brane. Yeast cells are having very tough cell wall because of this; there is very little effect of chemicals on yeast cells for disruption. Some enzymes act on the yeast cells to break open the content, but their application is not feasible for industrial purposes. We established the cell lysis by using the mechanical disintegrater which gives more than 85% of cell lysis in one shot. This cell lysate was subjected to different types of chaotropic agents in different concentrations to selectively leach out the membrane bound HBsAg. Each chaotropic agent showed different levels of effect during extraction. The highest protein quantity was recorded with sodium chloride and least was with urea. Further, the qual- ity of HBsAg extracted using urea was superior in comparison with other chaotropic agents. It was also found that the enrichment of HBsAg was found to be more in potassium thiocynate. In another experiment, the cells were exposed to the
KEY WORDS: HBSAG, H.POLYMORPHA, YEAST CELLS, CHAOTROPIC NON IONIC DETERGENTS
INTRODUCTION
During the early development of vaccine hepatitis B sur- face antigen,protein were purified from infected human plasma and used as a vaccine to prevent hepatitis B
ARTICLE INFORMATION:
*Corresponding Author: ziakhan7682@rediffmail.com Received 10th January, 2016
Accepted after revision 5th April, 2016 BBRC Print ISSN:
Thomson Reuters ISI SCI Indexed Journal NAAS Journal Score : 3.48
©A Society of Science and Nature Publication, 2016. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
virus infection. It was having several complications and threat of transferring other viral agents to the recipient and it too difficult to have mass vaccination because of limited supply of vaccine. To overcome these prob- lems the HBsAg genes have been cloned and expressed
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in E. coli, yeast cells and mammalian cells to reduce the dependency on plasma. The synthesis of HBsAg recovered as 22 nm particles in yeast cells (Hsieh et al. 1995), which is similar to the HBsAg isolated from the human plasma (Wijnendaele et al.1987; Wijnendaele et al. 1989, Pointek et al., 2000; Gerd Gellissen 2012).
HBsAg is a lipoprotein, the Escherichia coli expressed HBsAg was devoid off lipid portion so it was not use- ful for vaccination. To overcome this issue the gene for HBsAg was cloned into Yeast, it expressed the correct molecule but the protein was remained bound to the membrane. Yeast is surrounded by very thick and tough cell wall and its not easy to break open the way it is per- formed for bacteria and mammalian cells. Several meth- ods have been tried to break open the cell wall, (Zhang et
al., 1999; Wijnendaele et al., 1989 Pointek et al., 2000; Gerd Gellissen, 2012).
Traditional extraction techniques often involve harsh and extreme conditions like high pressure could potentially of damaging the desired products or limit yields. Enzymatic digestion breaks open the cell wall but its application is limited to the lab and its not feasible for industrial perspectives, (Knorr et al., 1979; Wijnen- daele et al., 1989). Furthermore, a second step is often required to break the spheroplasts generated from the commonly used digestive enzymes (zymolase, glucalase and/or lyticase). Sonication has been a widely used, suc- cessful method for cell disruption (Feliu et al. 1998) due to its speed, ease of operation, and cell lysis ability of various types, but until now it has only been relevant to
The most popular mechanical methods used for yeast cell lysis at a lab scale is to agitate the glass beads of 0.5mm diameter and cell suspension in a bead disinte- grator or simply on vortex mixer for
After disruption of the cells the next important objec- tive was to extract the protein of interest from the cell lysate suspension containing high percentage of impuri- ties.Chaotropic agents and
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cium and barium (Hurni et al., 1993; Craig et al., 1987).
The high speed centrifugation is found to be the most effective method to remove the cell debris from the homogenate suspension, apart from this many other methods like use of protein aggregating agents example
MATERIALS AND METHODS
Materials: All the raw materials used for fermentation and ER grade and the chemicals used in the purifica- tion were extra pure grade. These chemicals are from Merck Phosphate buffer: Phosphate buffer was prepared by adding 8.52 g Disodium hydrogen phosphate.12 H2O (Merck), 0.164 g Sodium dihydrogen phosphate. H2O (Merck) and 0.742 gm. EDTA into 90ml distilled water. Adjusted the final pH = 7.7±0.3 and final volume made up to 100 ml by adding distilled water.Cell lysis buffer: This was prepared by adding the triton
Fermentation: During the fermentations, H. polymorpha cells grows on synthetic glycerol based medium. Once, the cell density reached to the required optical density (OD)
Cell clarification/ separation: After the completion of induction process, the cells were subjected to the fil-
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Table 1: Chaotropic agents used for HBS Ag extraction
tration using the tangential flow filtration (TFF) 300kd NMWC. These cells further were washed three times with phosphate buffer to remove the fermented broth and lower the conductivity. At the end, the retentate is collected and diluted to required OD. This is starting material for cell breakage experiments. Alternatively for small scale preparations, 1000ml fermented broth (50- 60gm/L cell dry weight) was subjected for centrifuga- tion (6800 rcf for 10 min). Cell pellet was collected and washed twice with 1000ml of phosphate buffer by re- suspending the cell pellet and centrifuged at 6800 rcf for 10 min.
Cell disruptions: Two different types of methods were used for disrupting the yeast cells. 1. Cell disruption using the glass beads and vortex machine, and 2. Automated mechanical disintegrator (Dynomill KDL). For both the experiments, the starting material used was cell pellet suspension. Cell disruption by glass beads and overtaxing: The cell suspension was taken in a 50ml centrifuge tube and 7.5ml of glass beads were (0.5mm, Willy A.Bachhofen AG) added to the cell suspension.
The suspension was subjected for vortex for different timings and cell lysis was confirmed by microscopic observation.
Cell disruption Dyno Mill KDL: The cell pellet
Table 2: Effect of Triton
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Table 3: Percentage of cell disruption
Extraction of HBsAg from Cell lysate: using Chaotropic
agents: After establishing the required percentage of cell lysis, that is
EXTRACTION BY USING
In an another extraction experiment, the above cell lysate (70% broken cells) was treated with two
ferent concentrations to see the effect on the selective leaching of HBsAg from the lysed cells. The experiment is designed as mentioned in the table 2. Tube 1. The cell lysate was kept as such for incubation with out treating further, as a negative control for checking the HBsAg in the whole lysate
Tube 2. First the content of this tube was subjected for centrifugation, collected the pellet and supernatant separately and HBsAg was estimated in both (superna- tant and in pellet).
Tube 3. Taken whole cell lysate and added 0.1% Tri- ton
Tube 4 Taken whole cell lysate and added 0.1%
Tube 5, 6, 7, 8, 9, 10. Taken equal volume of cell lysate in six tubes, all six tubes were subjected to cen- trifugation, collected the supernatant in one container and pellets were kept separately for treating them with triton
These all above tubes were subjected for overtaxing and incubated in cold room for 16hrs for extraction with
Table 4: Effect of chaotropic agents on HBsAg extraction.
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LMW= low molecular weight markers, Lane 1: cell lysate Lane 2: Control supernatant
Lane 3: PEG and Sodium Chloride, Lane 4: Urea, Lane 5: Lithium chloride, Lane 6:
Potassium Iodide, Lane 7: Guanidine hydrochloride, Lane 8: Potassium thiocynate.
FIGURE 1.
continuous shaking on rocker platform. The centrifuga- tion was performed (either before or after the incubation period) at 6800g for 10 minutes to separate the superna- tant and collect the pellet. The volume of cell lysate in each tube was 1.5 ml.
RESULTS AND DISCUSSION
H.polymorpah cell disruption by Vortex: The cell lysis was performed by subjecting the yeast cells to mechani- cal agitation in the presence of glass beads (0.55mm) but it gave
Extraction of HBsAg from homogenate by chaotropic agents: For extracting the HBsAg,
were treated with different types of chaotropic agents and following results were found as per table no. 4
Extraction of HBsAg from cell lysate by
Cell disruption: Before establishing the cell lysis using dynomill, several experiments were conducted at small scale by playing with various parameters. The most important parameters were: the size of the glass beads (0.55, 75, 1.0mm), volume of glass beads, dry cell weight, flow rate of cell suspension feeding to the dynomill chamber and agitator speed. A comparative cell lysis study was also performed by treating the cells with tween 20 and without treatment. The data of initial experiments are not shown here which were considered
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FIGURE 2. Graphical presentation of effect of chaotropic agents on
HBsAg extraction
tial experiments, like glass beads
The purpose of cell lysis, is to have limited broken cells with maximum number of damage cells/damaged cell wall so that the chaotropic agents can act on them to selectively remove the membrane bound HBsAg. And at the same time addition of
While establishing the process, fixed all the above mentioned parameters and the flow rate of the cell sus- pension was changed with time and samples were col- lected and analyzed microscopically for the percent cell lysis. It was found that: 1. As the flow rate of feeding cell suspension increased from
Table 5: Effect of
1= Cell lysate , 2 = Supernatant from cell lysate , 3= Supernatant with 0.1% triton
=supernatant of sample 3 , 6 = supernatant of sample 4, 7= pellet plus 0.1%
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1= Cell lysate , 2 = Supernatant from cell lysate , 3= Supernatant with 0.1% triton
FIGURE 3. Effect of Triton
nitely have a negative effect onto the final yield. 4. In low flow rates as the cells are damaged too much, this excessive lysis is contributing to increased impurity pro- file and this posed trouble during product purification.
From the on line sample analysis, it was found that the feeding flow rate (100rpm) at dry cell density (40- 45mg/L), agitator speed (2000rpm), glass bead volume (600ml), glass bead diameter (0.5mm) gives optimum cell lysis with less number of broken cells, maximum number of damaged and very less number of cell debries. These all parameters were fixed for getting the consistent cell lysis
After establishing the cell lysis step, cell lysate was treated with different types of chaotropic agents. HBsAg was estimated in supernatant with Auszyme kit® and total quantity of extracted protein was estimated by using Folin lowery method (ref). All samples were also loaded onto the
figure 2) the same type results have also been confirmed by Craig et al. (1987).
HBsAg purity profile looks better in the sample treated with 5% PEG and 1000mM sodium chloride. This can be found in table 4. The PEG600 with Sodium chlo- ride was found to be the most effective with 4.97mg/ml of HCP and 0.01142% purity ratio (HBsAg/HCP) as com- pare to the 7.39mg/ml of HCP and 0.0098% purity ratio (HBsAg/HCP) of the control sample, Guanidine hydro- chloride and Lithium chloride shows similar profile with respect of HCP concentration (5.24mg/ml and 5.8mg/ml respectively) and % purity (HBsAg/HCP =0.01124%).
This result is supported by the earlier work of many researchers, (Craig et al. 1987;
A lot of HBsAg seems to be losing because of insuffi- cient extraction method. Most of the HBsAg is bound to the membrane and if its not extracted properly its going to be waste and at the end it will have negative effect on the over all product recovery. The presence of
First, cell lysate was treated with 0.1% Triton
Second, the supernatant from the control sample was treated with Triton
Third, the pellet was suspended into the 0.1%, 0.5% and 1.0% triton
This can also be explained that HBsAg remains in two forms such as immunoreactive which is detected in
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the supernatant and
CONCLUSION
Yeast is having several advantages over prokaryotic sys- tem for expressing heterologus proteins and vaccines. HBsAg is expressed and remained bounded into the membrane. To get the maximum yield it is good to have high expression but not necessary. At the same time one should have an efficient and robust purification scheme to recover maximum from available starting mate- rial. We established the cell lysis procedure using the dynomill, glass beads and agitation speed and dry cell weight. But this not the optimum if some one work very closely there is lot of scope to improve the cell lysis step. This step where one can have a very strong control to limit the loss of host cell proteins by limited cell lysis. if cell lysis is less then the final yield will definitely ham- per. If the more lysis the quality of final product will have some quality issues.
Uses of chaotropic agents didn’t improve the recovery this may be due to they are not suitable for membrane bound proteins. Sodium chloride in the presence of PEG gives very good results; a kinetics studied can be per- formed to enrich the HBsAg using different concentrations of Sodium chloride and PEG. This may help in pelleting out the host cell protein as much as possible which defi- nitely help in purification and it will have positive impact on quality of HBsAg. There is lot of scope for increas- ing the recovery by
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