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Capacity and diversity are extremely important to the quality of various phage display libraries. In this work, lambda phage-based in vitro package was applied to construct a filamentous phage display antibody library so as to enlarge its capacity and introduce more sequence diversity in the final library. In vivo recombination via Cre recombinase/lox sites was also exploited to create V(H)/V(L) combination diversity based on multivalent package of lambda phage packaging extracts on phagemid DNA concatemers. The library constructed with 10 microg concatenated phagemid DNA and ten vials of lambda phage packaging extracts was calculated to contain 1.40 x 10(10) independent clones. Higher capacity can be easily achieved when more materials are consumed. This strategy is somewhat more efficient than prior methods.

Two chimeric proteins have been constructed. One consists of four parts: a portion of the low molecular mass singlechain urokinase-type plasminogen activator (scu-PA-32K, residues 144-411), a 15-mer linker sequence, the C-terminal amino-acid sequence (residues 53-65) of hirudin (Hir), and an RGD sequence derived from the leech protein decorsin, i.e. scu-PA(32 k)-linker-Hir (residues 53-65)-RGD peptide. The other comprises two main segments: scu-PA(32 k) and hirudin into which RGDSP is inserted between its residues 33 and 34, i.e. hirudin (residues 1-33)-RGDSP-hirudin (residues 34-65)-scu-PA(32 k). These two chimeric genes were expressed in Escherichia coli, and the products were purified by Zn 2+ -chelating Sepharose 4B chromatography and benzamidine Sepharose 6B chromatography. Our results suggested that these two chimeric proteins not only had plasminogen-dependent fibrinolytic activity, but also possessed platelet aggregation inhibitory activity and antithrombin activity.Keywords: scu-PA; hirudin; RGD motif; fibrinolytic activity; antithrombin activity.Single-chain urokinase-type plasminogen activator (scu-PA), consisted of 411 amino acids with 12 disulfide bonds [1], can be cleaved by plasmin at the Lys158-Ile159 peptide bond and converted into two-chain urokinase-type plasminogen activator (tcu-PA), with higher activity [2]. Currently scu-PA and tcu-PA are both clinically used as efficient thrombolytic drugs, but it has been found that some adverse reactions may occur after dosing, such as bleeding complications and plateletmediated reocclusion [3]. A low molecular mass form of suc-PA lacking the amino acid 1-143 (scu-PA-32K) has been reported to have very similar enzymatic activity and thrombolytic potency as compared with those of intact scu-PA [3]. It has been used to construct a number of chimeric scu-PAs.Hirudin is the most potential native thrombin-specific inhibitor, as it acts on both circulating and clot-bound thrombin [4,5]. Its N-terminal domain binds to the active site of thrombin, whereas its C-terminal fragment covers the recognition site of fibrinogen [6]. Previous investigations have discovered that the adhesion of platelets to vessel walls and the activation and aggregation of platelets are extremely important in blood coagulation. On activated platelets, the binding of surface glycoprotein GPIIb-IIIa to fibrinogen mediates platelet aggregation. GPIIb-IIIa recognizes a conserved tripeptide Arg-Gly-Asp (RGD) sequence, which is found in all its ligands [7]. Peptides containing the RGD motif inhibit the binding of fibrinogen to GPIIb-IIIa on activated platelets, thus inhibit platelets aggregation [8]. Some leech proteins are also potential GPIIb-IIIa antagonists and platelet aggregation inhibitors [9]. Structural studies of these proteins have shown that the RGD sequence is located at the most exposed top of a mobile loop, joining two strands and protruding from the protein core [10,11].Therefore, we constructed two trifunctional chimeras that combine thrombolytic activity, antithrombin activi...

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