Continuous affinity-based selection: rapid screening and simultaneous amplification of bacterial surface-display libraries.

A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immobilized ligand of interest. Bacteria retained in this affinity fermenter were allowed to grow under continuous washout conditions, such that weakly bound organisms were selectively lost. Those initially rare founder bacteria expressing a receptor for the immobilized ligand (R+ve) were thus enriched and amplified simultaneously. From an initial culture containing 1 x 10(10) R-ve cells spiked with fewer than 30 R+ve bacteria (<1 in 10(8)), final ratios of R+ve/R-ve bacteria as high as 1 in 12 were observed, representing an enrichment factor of 55 million-fold. This technology has considerable potential for rapid screening of bacterial surface-display libraries and in facilitating directed-evolution studies.

Recent developments in heterologous protein production in Escherichia coli.

During the past three to four years, remarkable progress has been made in our understanding of protein folding, protein translocation across biological membranes, and the role of molecular chaperones in these processes. In conjunction with recent developments in Escherichia coli expression systems, this understanding has led to an improved capability to accumulate proteins in a soluble form, secrete proteins from the cell cytoplasm, accumulate proteins in the cytoplasmic membrane, and direct proteins to the outer membrane of the cell for surface display. These advances suggest that E. coli should now be considered seriously for applications that, only a few years ago, would have been thought beyond the scope of this organism.

Purification and characterization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli.

Plasminogen-activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-1/litre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37 degrees C with a half-life of approximately 48 h. At 20 degrees C, PAI-1 activity was stable for a week and at 4 degrees C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of high-level expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.

Functionally important conserved amino-acids in interferon-alpha 2 identified with analogues produced from synthetic genes.

A gene was chemically synthesised and expressed in Escherichia coli to produce [Ala30,32,33]IFN-alpha 2, an analogue of human alpha 2-interferon (IFN-alpha 2) which is devoid of activity on human cells. Eight additional analogues provided single changes in IFN-alpha 2 at each of these three conserved positions. No one residue is essential for activity, but both antiviral and anti-proliferative activity are particularly sensitive to changes in the side-chain of Arg33.

The isolation and characterization of three types of vitamin B6 auxotrophs of Escherichia coli K12.

Approximately 500 vitamin B6 auxotrophs were isolated from 18 independent cultures of Escherichia coli strain CR63. None grew in minimal medium supplemented with 2'-hydroxypyridoxine. Eighteen auxotrophs which had arisen independently were further characterized. All of them were defective in vitamin B6 synthesis rather than in an aminotransferase involved in vitamin B6 utilization. Two different phenotypes were recognized: 'Oxidase' mutants which grew only when supplied with pyridoxal or pyridoxal 5'-phosphate and 'Pre Pn' mutants which would also grow with pyridoxine or pyridoxine phosphate. "Oxidase' mutants were confined to a single linkage group, but data from interrupted mating experiments established that 'Pre Pn' mutants fall into two linkage groups which are possibly identical to pdxA and pdxB. All mutations in the in the pdxA region were allelic rather than located in two closely linked genes.

Synthesis of vitamin B6 by a mutant of Escherichia coli K12 and the action of 4'-deoxypyridoxine.

Mutants of Escherichia coli K12 blocked in the oxidation of pyridoxine 5'-phosphate ('Oxidase' mutants) excreted pyridoxine at an initial rate of 19 pmol h-1 (10(8) bacteria)-1, i.e.0.6 nmol h-1 (mg dry wt)-1, when starved for pyridoxal. Glycolaldehyde, L-phosphoserine, DL-serine and, to a lesser extent, L-leucine stimulated the rate of pyridoxine excretion, but there was no significant stimulation by 2'-hydroxypyridoxine. 4'-Deoxypyridoxine inhibited or stimulated growth of the "Oxidase' mutant, depending on the relative concentrations of added pyridoxal and 4'-deoxypyridoxine. It was concluded that stimulation of growth by 4'-deoxypyridoxine was due to its conversion to pyridoxal.