Human MAP2 Projection P3 Cat# Prot-r-MAP2AB-P3

      Microtubule associated protein 2 or MAP2 is a major microtubule binding protein of neurons, where it is concentrated in dendrites and perikarya but absent from axons (1). There is a single mammalian MAP2 gene which may generate multiple lower molecular weight forms usually named MAP2C and MAP2D which run on SDS-PAGE gels at 60-70kDa, though are actually much smaller in molecular size. These forms are found early in development but as the animal matures are replaced by MAP2A and B which are much larger in molecular size. These two forms include the so-called projection domain, a long insert which results in both molecules running at ~240kDa on SDS-PAGE gels. All four forms contain an N-terminal region which includes a binding site of cAMP dependent protein kinase (1).

      We inserted cDNAs encoding the various MAP2 sequences into the pET29a eukaryotic expression vector. The vector adds an C-terminal His-tag which was use to purify the protein and this, along with some other vector derived sequence, adds about 5 kDa to the molecule, underlined in the sequence below.. These sequences include an S-tag sequence shown in red, a thrombin cleavage site in green and the His tag in yellow. The MAP2 sequence is amino acids 1137-1588 from Uniprot P11137. This corresponds to the third segment of the “projection domain” a long insert expressed in MAP2A and MAP2B but absent from MAP2C and MAP2D. This insert is responsible for thin side arms seen on microtubules in neuronal dendrites. The construct was expressed in pET29a which adds a few N and C-terminal amino acids which are underlined below. The N terminus contains an S-tag, highlighted in red, and a thrombin cleavage site in green. The C-terminal His-tag, highlighted in blue, was used to purify the protein


MKETAAAKFE RQHMDSPDLG TLVPRGSMAD IGSEFSSKAP QEADAFMGVE SGHMKEGTKV
SETEVKEKVA KPDLVHQEAV DKEESYESSG EHESLTMESL KADEGKKETS PESSLIQDEI
AVKLSVEIPC PPAVSEADLA TDERADVQME FIQGPKEESK ETPDISITPS DVAEPLHETI
VSEPAEIQSE EEEIEAQGEY DKLLFRSDTL QITDLGVSGA REEFVETCPS EHKGVIESVV
TIEDDFITVV QTTTDEGESG SHSVRFAALE QPEVERRPSP HDEEEFEVEE AAEAQAEPKD
GSPEAPASPE REEVALSEYK TETYDDYKDE TTIDDSIMDA DSLWVDTQDD DRSIMTEQLE
TIPKEEKAEK EARRSSLEKH RKEKPFKTGR GRISTPERKV AKKEPSTVSR DEVRRKKAVY
KKAELAKKTE VQAHSPSRKF ILKPAIKYTR PTHLSCVKRK TTAAGGESAL APSVFKQAKD
KVSVDKLAAA LEHHHHHH


Number of amino acids: 498
Molecular weight: 55335.08
Theoretical pI: 4.79

Amino acid composition:
Ala (A) 45 9.0%
Arg (R) 22 4.4%
Asn (N) 0 0.0%
Asp (D) 35 7.0%
Cys (C) 3 0.6%
Gln (Q) 16 3.2%
Glu (E) 78 15.7%
Gly (G) 20 4.0%
His (H) 17 3.4%
Ile (I) 21 4.2%
Leu (L) 24 4.8%
Lys (K) 45 9.0%
Met (M) 9 1.8%
Phe (F) 12 2.4%
Pro (P) 29 5.8%
Ser (S) 47 9.4%
Thr (T) 33 6.6%
Trp (W) 1 0.2%
Tyr (Y) 7 1.4%
Val (V) 34 6.8%

Total number of negatively charged residues (Asp + Glu): 113
Total number of positively charged residues (Arg + Lys): 67

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 16055
Abs 0.1% (=1 g/l) 0.290, assuming all pairs of Cys residues form cystines

Ext. coefficient 15930
Abs 0.1% (=1 g/l) 0.288, assuming all Cys residues are reduced