Jingming Yuan

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain

PICK1 (protein interacting with Ckinase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn2+‐binding site. The Zn2+‐binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C‐terminal acidic region). Deleting the N‐terminal acidic region of NPDZ domain (involving PDZ domain and N‐terminal acidic region) in PICK1 impairs its Zn2+‐binding capacity.The mutation of the CPC (Cys‐Pro‐Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn2+‐binding. In addition, Zn2+ can enhance the lipid‐binding ability of PDZ domain as observed in both protein‐lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn2+ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.

Potential roles of recombinant acetylcholine receptor α subunit 1–211 in immunoadsorbent and DNA immunization

The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25 mg/L. rhAChR(α211) was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChR(α211) was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA-hAChR(α211). Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patients sera and establish an animal model of myasthenia gravis by recombinant hAChR(α211) immunization.