~What I have Done??~

1. I've got a clone called M15-pQEC1 (Escherichia coli M15 as a host, pQE-30 as expression vector that carried phaC1 gene). Then I continued with optimization of protein expression condition as below :

~Culture was grown first at 37°C, 180 rpm for 2 and a half hour until OD₆₀₀ reach 0.5 -0.6
(midexponential phase), before induction of 0.01 mM IPTG.

INCUBATION TEMPERATURE	INDUCTION PERIOD	SAMPLES COLLECTION
37°C	4 hours	Every hour
30°C	8 hours	Every 2 hours
28°C	4 hours	Every hour
24°C	12 hours	Every 3 hours
20°C	24 hours	Every 6 hours
15°C	24 hours	Every 6 hours

~Extraction of expressed total protein and also solubility test was done in order to determine at
which condition the protein expressed give more soluble form.

2. Co-transformation with chaperon plasmid, pGro7 ( Chloramphenicol for plasmid selection and
L-arabinose for induction of chaperone expression ) in order to improve protein solubility. I've
called this clone as M15-pQEC1/pGro7. Optimization of protein expression was done with same
condition as above.

~I found that induction at 28°C give more soluble form compared with other induction temperature.
While M15-pQEC1/pGro7 give better result than M15-pQEC1

3. Purification using Metal Affinity Chromatography - only done with soluble fraction of the protein.

4. Bovine Serum Albumin (BSA) Standard Curve was done for determination of protein
concentration . Based on the standard curve..I managed to get about 0.6 mg/ml protein.
This concentration of protein did not good enough for crystallization screening.



*Current work : Optimising the process to obtain sufficient sample to be screened for crystallisation conditions

p/s:I'll update more.......