Techniques update

Small-pool screening

A new technique called in vitro (a)

u

Unamplified 04 Pool expression cloning (h/EC) has been cDNA library #l #2 #3 developed in Marc Kirschner’s lab- ---

oratory at Harvard Medical School. Using this technique, cDNA li- Plate 100 colonies/plate

braries can be screened directly for proteins that have an interest-

ing biochemical or biological property. Theoretically, the tech- Scrape colonies

nique could be used in conjunc- 1 Prepare plasmid DNA

tion with any type of bioassay or in vitro biochemical assay.

In a traditional expression- cloning experiment, a portion of a cDNA library containing 1 03-1 OS clones is expressed and the re- sulting mixture of proteins is tested

uuuu Plasmid pools

i

Transcribe and translate with p5S]methionine

using a biological assay. The por-

tion of the library giving a posi- tive signal is subdivided until a mm

single active clone is isolated. This Protein pools

approach has been used success- FIGURE 1 fully to isolate genes encoding cytokines, ion channels, receptors

(a) Schematic diagram of the in vitro expression cloning (IVEC) technique. (b) Example of use of the technique to detect proteins degraded during

and developmental signalling mol- mitosis. Plasmid pools derived from a Xenopus cDNA library were translated in

ecules’. In each case, exquisitely the presence of [35S]methionine, producing about 25 distinct radioactive

sensitive assays were available to protein bands per pool. The crude translation mixture was mixed with

detect extremely small amounts of Xenopus egg extracts that had been arrested in either interphase or mitosis.

protein in the expression mixture. After a brief incubation, the two samples were loaded side-by-side on a

The IVEC procedure is similar in polyacrylamide gel, electrophoresed and analysed by autoradiography.

design, but the number of clones Proteins that were stable in the interphase extract (I) but degraded in the mitotic extract (M) were identified, and their cDNA clones were isolated from

in each pool to be tested is much the original pool by sib selection. The arrowhead indicates a protein degraded

smaller, typically of the order of during mitosis. From 50 000 clones, this screen identified B-type cyclins, which

100 (Fig. 1). The small size of the are known to be degraded during mitosis, and one new protein.

pool offers three key advantages. First, the amount of each protein produced is pro- for the protein is particularly abundant. A cDNA portionally greater, so that less-sensitive assays are that is scarce or poorly translated in vitro would be sufficient to detect the clone of interest in the mix- difficult to detect. ture. Second, the amount of subdivision needed to

isolate a single clone is much less. Third, the number References of proteins produced is small enough that they can 1 SIMONSEN, H. and LODISH, H. F. (1994) Trends

be analysed on polyacrylamide gels. This allows the Pharmocol. Sci. 15, 437

identification of proteins that are modified or de- 2 LUSTIC, K. D. et 01. Methods Enzymol. (in press)

graded under certain conditions. 3 STUKENBERC, P. T., LUSTIC, K. D., McGARRY, T. J.,

Variations of the IVEC technique have already been KING, R. W., KUANC, 1. and KIRSCHNER, M. W. (1997)

used to isolate cDNA clones for many different types Curr. Biol. 7, 338-348

of protein9. These include proteins phosphorylated 4 LUSTIC, K. D., KROLL, K. L., SUN, E. E. and

during mitosis3, proteins clipped by caspases during KIRSCHNER, M. W. (1996) Development 122, 4001-4012

apoptosis, developmental signalling molecules4, and

DNA-binding proteins. The technique seems to be Thomas McCarry, Dept of Cell Biology, Harvard Medical

most successful when there are many proteins in the School, 240 Longwood Avenue, Boston, MA 02115, USA.

library that could give a positive signal or if the cDNA E-mail: tmcgarry@warren.med.harvard.edu

374 Copyright 0 1997 Elsevier Science Ltd. All rights reserved. 0962-8924/97/$17.00

PII: SO962.8924(97)01131-S

trends in CELL BIOLOGY (Vol. 7) September 1997