~Progress 2~

I already reculture M15-pQEC1/pGro7 and M15-pQEC1 in 400 ml LB at 28°C and also the induction was also at the same temperature.

Purification was done. All the elution give approximately a band at around 60 kDa.

Based on the standard curve..I managed to get about 0.35 mg/ml of protein for M15-pQEC1/pGro7 and 0.2 mg/ml of protein for M15-pQEC1 .

I'm thinking of...if from 400 ml of culture, I get the amount shown above..

Theoritically, I'll get higher concentration of protein from large amount of culture.

Thus, I'm trying to produce more protein from the 2.0 L bacterial cultures.

*Since M15-pQEC1/pGro7 give better result than M15-pQEC1..therefore I've decided to proceed the optimisation process by using M15-pQEC1/pGro7.

p/s : I'll update the result soon........

~future work: concentrate the protein and trying to redo gel filtration

~Progress~

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

Thus, my attempt is to grow the cell at 28°C until it reach OD600 reach 0.5 -0.6
(midexponential phase),and also the induction was also at the same temperature.
*to promote slow grow of cell so that the cell was not force to express the protein, and allow the protein to fold properly in order to get higher amount of protein.

Results:
1)I have done solubility test and also purification. All the elution give approximately a thick band at around 60 kDa.Its indicate that my protein is quite a lot.

2)Since there's a lot of contaminants, I've decided to do gel filtration (size -exclusion chromatography)to get purity of protein.

Problem:

~There's no peak of elution appear after filtration was done.I redo the filtration but I got the same results. Where is my protein????

Troubleshoots:

~ May be my protein precipitate in low concentration of salt. This is because, I notice that [NaCl] in the buffer use for filtration was lower compared to the one in the buffer used for Affinity Chromtography.It's my mistake..
*Redo gel filtration with the same and/or higher [NaCl] in the equilibration/wash buffer

~ My protein was degrade since it's a week olds.
* Rerun the SDS-PAGE to check the stability of the protein. I've noticed that my protein which supposed to have MW of 60 kDa was now at ~30-40 kDa. I think that's why I've got negative result for gel filtration.


-----I'll culture, expressed, and purify the protein straight away...Hopefully I'll success in my work-----

~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.......

Overview of My Research

Polyhydroxyalkanoates (PHAs) are produced by a wide variety of bacteria as an intracellular storage material of carbon and energy. PHA synthase (PhaC) is the key enzyme in the production of PHAs.Basically, my project involve this enzyme from Pseudomonas sp. USM4-55. It is a Gram negative bacterium that is capable of accumulating short and medium chain length polymer. phaC1 and phaC2 genes of Pseudomonas sp. USM4-55 have already been cloned.The phaC1 gene was subcloned again to optimize soluble protein production in Escherichia coli. Proteins was then purified with the help of affinity tags.Currently I'm optimizing the process to obtain sufficient sample to be screened for crystallization conditions with the goal of single crystal X-ray diffraction.

* Aim : A single crystal for structure prediction..InsyaALLAH~