cloning, protein expression and purification

Cloning

Cloning of genes and bacteria

Escherichia coli Rosetta-gami2(DE3) selected as bacterial host for this cloning.

Expression vector pET32 Xa/Lic used as expression vector.

PHA synthase from Chromobacterium spp. USM2 was amplified by PCR and cloned into expression vector and transformed into bacterial host.

The culture conditions for E.coli RG2 (DE3) pET32-phaCcs are 37degree C, shake 180 rpm.

For selection indicator, This transformant resist Chloramphenicol, Streptomycin, tetracycline and Ampicillin.

Protein Expression

Induction

Transformed bacteria were cultured at 37degree C, 180 rpm for 4-4hours 30mins to reach an OD600nm reading of ~0.5.

Then, a concentration of 0.4mM IPTG added into the culture.

The bacteria suspension were incubate at 30degree C, 180rpm for 8hours.

Sonication

Cells were break by sonicate @ 90V power output for 1 minute and cold for 1minute (5cycles).

Then, centrifugation at 4degreeC kubota at 8000rpm, 20minutes to pellet the insoluble fraction protein.

The supernatant was treated as soluble fraction protein.

SDS P.A.G.E

The uninduced total protein, induced total protein, Soluble fraction, Insoluble Fraction and protein ladder were used to run SDS P.A.G.E for analysis.

Purification

Metal affinity chromatography

Only the soluble fraction of the protein were proceeded with purification.

SDS P.A.G.E

The elution, binding flow through, washing flow through are used as SDS sample to analyse the purity.

Size exclusion chromatography

To remove Imidazole, further polishing the purity of the phaC protein.