Abstract
The CD4 cell surface antigen is of interest as a marker of T lymphocytes that recognize foreign antigens in the context of MHC Class II antigen, as a receptor for the human immunodeficiency virus (HIV) and as a member of the immunoglobulin superfamily (IgSF) with four Ig-like domains present in the extracellular domain. In order to produce large amounts of soluble CD4 for x-ray crystallography and other molecular studies, a recently developed expression system based on selection via glutamine synthetase was used. Expression was attempted for rat CD4 corresponding to the full extracellular sequence (sCD4; domains 1-4), the NH2-terminal half (domains 1 and 2) and the first domain alone. Stable transfected Chinese hamster ovary cell lines were obtained that expressed sCD4 and sCD4 (half) at typical maximal levels in spent tissue culture supernatant of greater than 80 and 25 mg/liter, respectively. Domain 1 alone was not expressed and introduction of a N-linked glycosylation site did not facilitate expression. The role of glycosylation in the expression of sCD4 was investigated by mutagenesis of the constructs to remove each of the two N-linked glycosylation sites in turn and both together. All three forms were expressed at 60-120 mg/liter. The sCD4 (half) was not expressed after deletion of its N-linked site. The disulfide bonds of sCD4 were determined to be within domains 1, 2, and 4 and isolation of glycopeptides showed that both N-linked sites were glycosylated. Analysis of the hydrodynamic properties of sCD4 suggested that the molecule adopted an extended conformation in solution rather than folding to form a compact structure like an Fab. The possibility of dimerisation of CD4 was investigated but sCD4 dimers were not seen at an affinity cut-off of about 4 x 10(5) M-1.
Highlights
High Level Expression in Chinese Hamster Ovary Cells of Soluble Forms of CD4 T Lymphocyte Glycoprotein Including Glycosylation Variants*
We report the production of soluble forms of rat CD4 using the glutamine synthetase-based expression system
These constructs were inserted into the pEEG.HCMV vector and the glutamine synthetase minigene was subcloned into the BanHI site (Fig. 1C)
Summary
Affinity Chromatography-The sCD4 and variant forms were purified from spent tissue culture medium by affinity chromatography and gel filtration (Clark et al, 1987; detailed methods are described by Arvieux and Williams, 1988). Spent tissue culture medium was passed through affinity columns containing 20 ml of Sepharose CL4B beads with W3/25, 0X35, or OX37 mAbs (Jefferies et al, 1985). M Tris-HCl, pH 7.4, and further purified by S-200 gel filtration as described above. 8, containing (a) 0.05 mg/ml [YS]sCD4 or (5) 0.05 mg/ml [?S]sCD4 and 1 mg/ml unlabeled sCD4 or (c) 0.05 mg/ml [3SS]sCD4 and 0.2 mg/ml W3/25 Fab was layered onto the gradients followed by 0.2 ml of 10 mM Tris-HCl, 150 mM NaCl, pH 8, alone or with 1 mg/ml unlabeled sCD4. The elution position of the [35S]sCD4 was determined by scintillation counting
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