Production of a recombinant C8[alpha] protein fragment for structural analysis. (South Carolina Academy of Sciences Abstracts).
C8[alpha] is a 64 kDa subunit of human complement components C8,
which is one of five components (C5b, C6, C7, C8, C9) that interact to
form the cytolytic membrane attack complex of complement (MAC).
C8[alpha] is homologous to the C8[beta] subunit and long with C6, C7,
and C9 form the MAC protein family. The objective of this project is to
produce and crystallize a recombinant form of the putative
membrane-binding domain of C8[alpha]. This self-folding domain is
referred to as the MACPF region. It is approximately 43 kDa in size,
contains two disulfide bonds and no carbohydrate. Recombinant MACPF has
been produced in small quantities in the mammalian cell line COS-7.
Experiments described here will show how cDNA clones encoding the
C8[alpha] MACPF were constructed and used to explore expression in a
bacterial system as a means of producing milligram quantities of
protein. Once expression has been optimized, the protein will be
purified and attempts will be made to obtain diffraction quality
crystals for structural analysis. This would be the first structural
information about a MAC family protein, thus providing new insight into
how C8[alpha] and its structural homologues (C6, C7, C8[beta], and C9)
perform their cytolytic function. (Supported by a SC-BRIN/EPSCoR CRP grant.)
Devon Bork, K. Murphy, B. Chiswell, James Sodetz *, Chasta Parker
Department of Chemistry
* Department of Chemistry and Biochemistry
University of South Carolina