High yielding recombinant Staphylokinase in bacterial expression system--cloning, expression, purification and activity studies.
Staphylokinase (SAK) is emerging as an important thrombolytic agent. In this report, we describe the cloning, expression, purification and activity studies of the SAK gene of Staphylococcus aureus from a custom synthesised SAK gene. The SAK gene of 411 bp yielded a protein of approximately 15 kDa when expressed under pET21a vector using IPTG as an inducer in BL21 (DE3) pLysE codon Plus cells. The recombinant SAK (rSAK) was soluble in nature and constituted nearly 35% of the total cellular protein as estimated by densitometry scanning. Fermentation studies were carried out to optimize various parameters for maximizing the yield of rSAK and with the optimized medium, the yield of rSAK was nearly 2.8 g/L of fermentation broth, which is highest yield of rSAK expressed in any bacterial system till date. Two simple purification steps of ion-exchange chromatography yielded homogenous rSAK with almost 36% recovery. The purified SAK protein was characterized by MALDI-TOF and by plasminogen activation studies. The rSAK was found to be active by the chromogenic substrate assay method.
Mandi
Nagnath
N
Biotechnology R & D, Lupin Limited, 46A/47A, Nande Village, Mulshi Taluka, Lupin Research Park, Pune-411042, India. nagnathmandi@lupinpharma.com
Soorapaneni
Sudheerbabu
S
Rewanwar
Sachin
S
Kotwal
Prakash
P
Prasad
Bhaskarjyoti
B
Mandal
Goutam
G
Padmanabhan
Sriram
S
D020029:Base Pairing; D001483:Base Sequence; D019149:Bioreactors / Q000382:microbiology; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013211:Staphylococcus aureus / Q000201:enzymology*
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D010958:Plasminogen; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus