ABSTRACT
Bacterial expression of full length beta-amyloid (Abeta) 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 semisynthetic method in which Abeta1-29 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-Abeta1-29-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-Abeta1-29-MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Abeta1-29-MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH2-A30C-Abeta30-40, which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Abeta1-29-MESNA. Native chemical ligation proceeds rapidly and efficiently (>90% yield) to give A30C-Abeta1-40. The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (>90% yield) to give native sequence Abeta1-40. Overall, this system is highly efficient, and can yield approximately 8-10 mg of pure Abeta1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Abeta peptides. We have also expressed an Abeta construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Abeta1-29-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 15N or 13C labels for NMR. Future work will also include extending this system to longer Abeta peptides, such as Abeta1-42.
Subject(s)
Amyloid beta-Peptides/biosynthesis , Amyloid beta-Peptides/chemical synthesis , Peptides/chemical synthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Chitin/chemistry , Chitin/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Peptides/chemistry , Peptides/genetics , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
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.
