BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.

There are many inhibitors of amylases.1~13) They are useful tools for investigation of the active site of amylases and the differences between amylases of various origins. On the other hand, the measurement of serum and urine amylase has clinical significance for diagnosis of diseases. For this purpose, an amylase inhibitor could be used for determination of activities of amylase isozyme.14) Immobilized amylase inhibitors are also effective for purification of amylases.15'16) During screening of amylase inhibitors of microbial origin, we isolated an inhibitor which inhibited pig pancreatic a-amylase strongly. The a-amylase inhibitor was designated as Paim (Pig pancreatic a-amylase inhibitor of microbes). This paper describes the purification and some properties of Paim. The assay system for Paim used was similar to that reported previously,2) except that pig pancreatic a-amylase was used instead of bac- The purification procedure for Paim II was almost the same as that for Paim I. The final inhibitor preparations were submitted to polyacrylamide gel disc electrophoresis at pH 2.3 and pH 9.5. A single band was obtained for Paim I, but Paim II consisted of one major band together with a few minor bands. Effects ofPaim I and II on various amylases were tested and the results are shown in Table I. Paim I and II showed inhibitory activity toward pancreatic a-amylase of pig, dog, cow and horse. Paim I and II showed no activity toward a-amylases of plant and microbial origins. It is interesting that pancreatic aamylases of pig, dog, cow and horse were inhibited by Paim I and II, but a-amylases of humans were not. These facts suggest that there are some structural differences between these amylases. Paim may be a useful tool

Paim I, a protein alpha-amylase inhibitor, is a single-chain polypeptide which consists of 73 amino acids, including 4 half-cystine residues. The positions of disulphide bonds in Paim I have been determined with the combination of enzymatic digestion and fast atom bombardment (FAB) mass spectrometry. Denatured Paim I was digested to peptides with Staphylococcus aureus V8 protease. These peptides were subjected to FAB mass spectrometry, with or without isolation by high-performance liquid chromatography. The positions of disulphide bonds in Paim I were determined from the relative molecular masses of the peptides containing a disulphide bond and by the enzyme specificity of S. aureus V8 protease. It is deduced that Paim I has two disulphide bridges at Cys(8)--Cys(24) and Cys(42)--Cys(70).