Recombinant Green Fluorescent Protein Cat# Prot-r-AcGFP

      The green fluorescent protein (GFP) is a 27kDa protein isolated originally from the jellyfish Aequoria victoria. It has an endogenous fluorochrome activity with excitation maximum at 395nm and emission maximum at 509nm, which is similar to that of fluorescein (1,2). The GFP gene was cloned and sequenced and the origin of the fluorochrome by autocatalytic activity of certain amino acids was discovered (3,4). Much interest in GFP was generated when it was shown that fluorescence develops rapidly when the protein is expressed and requires only molecular oxygen and no other cofactors. As a result GFP can be expressed in fluorescent form in essentially any prokaryotic or eukaryotic cell (5). GFP has been engineered to produce a vast number of variously colored mutants including blue, cyan and yellow protein derivatives, BFP, CFP and YFP (6-9). GFP and other fluorescent proteins derived from jellyfish, coral and other Cnidaria are widely used as tracers in transfection and transgenic experiments to monitor gene expression and protein localization in vivo and in in vitro. The crystal structure of GFP was determined (7) which allowed amino acid modifications to improve spectral properties and prevent multimerization (8,9). The 2008 Nobel prize in chemistry was awarded “for the discovery and development of the green fluorescent protein, GFP”.

Sequence taken from AY233272, download here, also Uniprot entry Q6YGZ0. This antibody was made against a recombinant construct expressed in and purified from E. coli using the pET29a (+) vector. This vector adds a few N and C-terminal amino acids which are underlined below. The N terminus contains an S-tag, highlighted in red below, which can be used to purify the protein. However we used the C-terminal His-tag (green below) to purify the protein. The GFP sequence is identical to that found in a series of widely used expression vectors.


MKETAAAKFE RQHMDSPDLG TLVPRGSMAD IGSEFMVSKG AELFTGIVPI LIELNGDVNG  60
HKFSVSGEGE GDATYGKLTL KFICTTGKLP VPWPTLVTTL SYGVQCFSRY PDHMKQHDFF 120
KSAMPEGYIQ ERTIFFEDDG NYKSRAEVKF EGDTLVNRIE LTGTDFKEDG NILGNKMEYN 189
YNAHNVYIMT DKAKNGIKVN FKIRHNIEDG SVQLADHYQQ NTPIGDGPVL LPDNHYLSTQ 240
SALSKDPNEK RDHMIYFGFV TAAAITHGMD ELYKVDKLAAALE HHHHHH 289

Number of amino acids: 289
Molecular weight: 32414.49
Theoretical pI: 5.78

Amino acid composition:
Ala (A) 18 6.2%
Arg (R) 8 2.8%
Asn (N) 15 5.2%
Asp (D) 21 7.3%
Cys (C) 2 0.7%
Gln (Q) 8 2.8%
Glu (E) 19 6.6%
Gly (G) 25 8.7%
His (H) 16 5.5%
Ile (I) 16 5.5%
Leu (L) 21 7.3%
Lys (K) 21 7.3%
Met (M) 10 3.5%
Phe (F) 15 5.2%
Pro (P) 12 4.2%
Ser (S) 14 4.8%
Thr (T) 19 6.6%
Trp (W) 1 0.3%
Tyr (Y) 12 4.2%
Val (V) 16 5.5%


Total number of negatively charged residues (Asp + Glu): 40
Total number of positively charged residues (Arg + Lys): 29

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 23505
Abs 0.1% (=1 g/l) 0.725, assuming all pairs of Cys residues form cystines

Ext. coefficient 23380
Abs 0.1% (=1 g/l) 0.721, assuming all Cys residues are reduced