Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

Bacterial expression of full length β-amyloid (Aβ) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semi-synthetic method in which Aβ 1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Aβ 1-29 -Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Aβ 1-29 -MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Aβ 1-29 -MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH 2 -A30C-Aβ 30-40 , which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Aβ 1-29 -MESNA. Native chemical ligation proceeds rapidly and efficiently (> 90% yield) to give A30C-Aβ 1-40 . The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (> 90% yield) to give native sequence Aβ 1-40 . Overall, this system is highly efficient, and can yield ≈ 8-10 mg of pure Aβ 1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Aβ peptides. We have also expressed an Aβ construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Aβ 1-29 -Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15 N or 13 C labels for NMR. Future work will also include extending this system to longer Aβ peptides, such as Aβ 1-42 .