MAP2-P3 Protein

MAP2-P3 Protein


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 have expressed five different recombinant forms of MAP2 based on the human sequence reported in REFSEQ XP_006712595.. These constructs in combination cover almost the entire human MAP2 molecule. We have used these constructs to generate a series of monoclonal and polyclonal antibodies to MAP2, many of which are known to recognize defined segments of MAP2.

MAP2 Proteins
The S lane shows molecular weight standards of the indicated apparent molecular weights in kiloDaltons. The next 5 lanes show 2µg amounts of purified recombinant forms of various human MAP2 proteins. D is recombinant full length MAP2D and C is recombinant full length MAP2C. MAP2D is 559 amino acids while MAP2C is 471, missing one of the microtubule binding domains of MAP2D and a more N-terminal 57 amino acid N-terminal sequence. Lanes P1, P2 and P3 show each recombinant segments of the human MAP2 projection domain, found in MAP2A and B but absent from MAP2C and D. The P1 construct is amino acids 233-684, the P2 is 712-1136 and P3 is 1137- 1588. All of the constructs run more slowly on SDS-PAGE than expected from their molecular size, probably due to their high content of charged amino acids. These constructs were used to make a series of mouse monoclonal antibodies. The lane marked BSA contains 2µg of bovine serum albumin, another gel standard.

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.

The sequence is amino acids 1137-1538 from REFSEQ XP_006712595. This corresponds to the the third segment of the “projection domain” found in MAP2A and MAP2B but missing from MAP2C and MAP2D. The construct was expressed in pET29a and has a few N and C-terminal amino acids derived from the vector, including a C-terminal His-tag.

>MAP2-P3
SSKAPQEADAFMGVESGHMKEGTKVSETEVKEKVAKPDLVHQEAVDKEESYESSGEHESL
TMESLKADEGKKETSPESSLIQDEIAVKLSVEIPCPPAVSEADLATDERADVQMEFIQGP
KEESKETPDISITPSDVAEPLHETIVSEPAEIQSEEEEIEAQGEYDKLLFRSDTLQITDL
GVSGAREEFVETCPSEHKGVIESVVTIEDDFITVVQTTTDEGESGSHSVRFAALEQPEVE
RRPSPHDEEEFEVEEAAEAQAEPKDGSPEAPASPEREEVALSEYKTETYDDYKDETTIDD
SIMDADSLWVDTQDDDRSIMTEQLETIPKEEKAEKEARRSSLEKHRKEKPFKTGRGRIST
PERKVAKKEPSTVSRDEVRRKKAVYKKAELAKKTEVQAHSPSRKFILKPAIKYTRPTHLS
CVKRKTTAAGGESALAPSVFKQAKDKVS


References:

1. Dehmelt1, H and Halpain, S. The MAP2/Tau family of microtubule-associated proteins.
Genome Biol. 204 (2005)

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