1) Cultivation of E.coli W31101Q
- truncated gene was streak
- incubated 24 hr & transfer single colony
2) Fermentation
- Incubate at 37ºC,( 200, 250 or 275 )rpm for 18 hours.
3) Harvesting
- centrifuge at 6100 rpm for 10 min at 4°C
- collection of pellet
4)cell disruption
- chemical lysis, ultrasonic bath, enzymatic lysis
5) Precipitation
- precipitate with ammonium sulphate (35% saturation)
6)Dialysis
- using dialysis tube & dialysis overnight
7)Determination of HBcAg
- 15% polyacrylamide
- staining using comassie blue
Methodology
Cultivation of E.coli W31101Q
Literature review
The core particle has become one of the most
frequently studied systems as a carrier for various foreign epitopes (Touze et al., 1999)
E.coli chosen as an effective gene expression and satisfactory production of recombinant protein (Beng et al., 2004)
comparative evaluation of different rotational speed of HBcAg
Expanded bed adsorption chromatography (EBA) has been developed widely in the purification and allows direct purification of the bio-products from unclarified feedstock (Michelle et al., 2007)
Determine HBcAg by using SDS-PAGE
Chemical lysis
Optimum condition for Hepatitis B core antigen production in shaked flask fermentation
- higher yield produce at 200 rpm
- no significantly change at speed of 250 and 275 rpm
Result & Discussion
- hepadnaviridae family
- nucleocapsid consists of 180 to 240 subunits of core protein
- highest in sub saharan Africa and East Asia
- used widely as a nanocarrier for the development of multicomponent vaccines due to its self assembly and antigenic properties (whitacre, 2009)
- used BugBuster, protein extraction reagents, protein extraction reagens
Ultrasonicator bath & enzmatic lysis
Introduction
Objectives
- First, performed optimization of rotational speed of 200, 250 and 275 rpm at constant temperature of 37 °C
- Second, performance of several cell disruption techniques such as ultrasonification bath, enzymatic lysis and chemical lysis by using optimum rotational speed at 200 rpm
- wide range protein molecular weight prestained marker, range of 10- 170 kDa
- obtained band 17 kDa
Komathi a/p sandarammutee
55247211146
- band formed, thus can proceed to dialysis
- To determine the optimum rotational speed for Hepatitis B core antigen production in shake flask fermentation
- Comparative evaluation of different cell disruption method for the release of recombinant Hepatitis B core antigen from Escherichia coli
Hepatitis B virus
- most serious types of viral hepatitis
- discovered in 1965 when Australia antigen was found
- causes cirrhosis and liver cancer
- more than 360 million chronic HBV and annual mortality about 2 million (Shepard, 2013)
- until now there is no specific treatment for this disease
- Ultrasonic bath produce higher yield
- Ultrasonificator bath is usually used for degassing function to remove small bubbles from liquid
- See Seiter,the use of an ultrasonic bath to disrupt cells such as Mycobacterium tuberculosis in a sample to which beads of glass or other materials in the range of 50 microns to 1mm have been added. Following disruption released RNA and DNA into solution for hybridization with genetic probes
Comparative evaluation of different plasmid of HBcAg
Oligomerization state
Importance to produce purified HBcAg
Why need to find optimum condition and method
- band form at the truncated were thicker compared to his-tag.
- The yield of this truncated HBcAg derivative in E.coli is considerably higher than His-tag HBcAg expressed from equivalent plasmid constructions.
Important to produce
high yield of HBcAg
at present stage
- seen that the dimer form thicker band compared to monomer
- trimer and tetramer cannot be seen clearly.
- That optimum growing parameter for E.coli is 200 rpm at 37 °C.
- Ultrasonicator bath is suitable methods for rupturing E.coli cell walls
- The higher yield present in dimer cause of the natural dimeric structural of the truncated HBcAg compared to monomer.
- The yield of this truncated HBcAg derivative in E. coli is considerably higher than the His-tag HBcAg expressed from equivalent plasmid constructions.
- Important to develop a rapid and simplified recovery and purification method for recombinant HBcAg in order to achieve a better overall yield and purity
- production could be scale up into a bioreactor to determine other parameters that influences the fermentation process.