MC LACHUN. BERNSTEIN. AND ANDERSON 
FIG. 2. Elution profiles of cell lysates separated by FPL-ion-exchange chromatography. A Mono Q 
column (HR 5/5) w « equilibrated with 0.05 M KG, 20 dm Tris-HG buffer, pH 7.5 (Buffer A). The 
clarified cell extract was loaded on the column in Buffer A in a volume of 500 at a flow rate of 0.5 
ml/ min. The column was washed with 10 ml of Buffer A followed by the linear KG gradient shown. 
Fractions with a volume of 1.0 ml were collected and assayed for ADA activity. The upper protein profile 
(A) is a lysate from NIH mouse 3T3 cells infected with the SAX vector, and the lower protein profile (B) is 
a lysate from monkey OM cells infected with the SAX vector. 
the human ADA cDNA using the SAX ret- 
roviral vector. A total cell lysate was sepa- 
rated on the Mono Q column (Fig. 2B), and 
paired fractions were analyzed by starch gel 
electrophoresis (Fig. 4). Human ADA iso- 
zymes eluted from the column in fractions 
3-6, whereas typical endogenous monkey 
isozymes were visible in fractions 17-20. 
CEM cells and uninfected CV-1 cells were 
run as human and monkey cell controls on 
the same gel. These results show that the 
human ADA protein produced by the SAX 
vector in CV-l cells has the characteristic 
human isozyme pattern and can be separated 
from the endogenous monkey ADA on a 
Mono Q column. Similar studies have dem- 
onstrated the separation of human from 
monkey ADA in the lysates of cynomolgus 
macaque cultured T cells that were success- 
fully infected in vitro with the SAX vector 
(data not shown). Therefore, the human 
ADA isozyme pattern observed after transfer 
of the human ADA gene is the same whether 
the host cell is from a low-expressing (kid- 
ney) or high-expressing (lymphocyte) tissue. 
Also, when a cell extract from a mixture of 
human CEM cells and peripheral blood 
mononuclear cells from the monkey is frac- 
tionated on a Mono Q column, the same sep- 
aration of human and monkey ADA en- 
zymes is observed (data not shown). 
The method outlined in this paper de- 
scribes the separation of human ADA, intro- 
duced by a retroviral vector, from mouse or 
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Recombinant DNA Research, Volume 12 
