Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli

Maywald, Friedhelm; Böldicke, Thomas; Groß, Gerhard; Frank, Ronald; Blöcker, Helmut; Meyerhans, Andreas; Schwellnus, Konrad; Ebbers, Jürgen; Bruns, W.; Reinhardt, G.; Schnabel, Eugen; Schröder, Werner; Fritz, Hans; Collins, John

doi:10.1016/0378-1119(88)90038-8

Cited by 23 publications

(9 citation statements)

References 38 publications

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“…In view of this knowledge the human PSTI which inhibits strongly its natural antagonist, the pancreatic trypsin, was chosen for a protein design project. 36 Our aim was to develop inhibitors with high affinity against human neutrophil The primary structure of PSTI and its subsite positions (residues) in most intimate contact with the target enzyme(s) in the complex are shown in figure 3. The primary inhibitory specificity of various artificial mutants of PSTI produced by recombinant DNA techniques is indicated in table IV.…”

Section: Kazal-type Inhibitorsmentioning

confidence: 99%

Proteinase Inhibitor Candidates for Therapy of Enzyme-Inhibitor Imbalances

Fritz

Collins²,

Jochum

1992

Biochemistry of Pulmonary Emphysema

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Section: Kazal-type Inhibitorsmentioning

confidence: 99%

Proteinase Inhibitor Candidates for Therapy of Enzyme-Inhibitor Imbalances

Fritz

Collins²,

Jochum

1992

Biochemistry of Pulmonary Emphysema

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“…PSTI 0 was purified as described by Maywald et al [2]. For affinity chromatography of PSTI 4 a chymotrypsin-Sepharose column was used.…”

Section: Purification Of Pstimentioning

confidence: 99%

“…Blotting was carried out as described by separate antibody-bound inhibitor from free inhibitor. Incu-Maywald et al [2]. In order to raise antibodies to small peptides, the peptides are usually coupled to the following carriers : bovine serum albumin and hemocyanin [18], ovalbumin [19], thyroglobulin and fibrinogen [20].…”

Section: Radioimniunoassay Procedures Determination Of the Subclasses mentioning

confidence: 99%

“…The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.The genes of human pancreatic secretory trypsin inhibitor (PSTI 0, [l]) and a variant (PSTI 4) of this wild-type sequence were chemically synthesized, cloned and expressed by a secretion vector in Escherichia coli [2]. It was shown that the substitution of Lys-18, Ile-19 and Asn-21 in PSTI 0 by Leu-18, Glu-19 and Arg-21 in PSTI 4 (Fig.…”

mentioning

confidence: 99%

“…The genes of human pancreatic secretory trypsin inhibitor (PSTI 0, [l]) and a variant (PSTI 4) of this wild-type sequence were chemically synthesized, cloned and expressed by a secretion vector in Escherichia coli [2]. It was shown that the substitution of Lys-18, Ile-19 and Asn-21 in PSTI 0 by Leu-18, Glu-19 and Arg-21 in PSTI 4 ( Fig.…”

mentioning

confidence: 99%

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Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides

Böldicke¹,

Kindt²,

Maywald³

et al. 1988

European Journal of Biochemistry

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Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.The genes of human pancreatic secretory trypsin inhibitor (PSTI 0, [l]) and a variant (PSTI 4) of this wild-type sequence were chemically synthesized, cloned and expressed by a secretion vector in Escherichia coli [2]. It was shown that the substitution of Lys-18, Ile-19 and Asn-21 in PSTI 0 by Leu-18, Glu-19 and Arg-21 in PSTI 4 (Fig. 1) led to a distinct alteration of the specificity of the protease inhibitor. While PSTI 0 inhibits human trypsin (Ki = 1.5 x M, PSTI 4 inhibits human granulocyte elastase (Ki = 6 x lo-" M) and chymotrypsin (Ki < 5 x lo-" M).For clinical diagnostic purposes we aimed at producing specific monoclonal antibodies against the active sites of PSTI 0 and PSTI 4. These should allow the determination of the amount of free protease inhibitor in the presence of bound inhibitor in serum. To produce monoclonal antibodies that are directed against the active sites of PSTI 0 and PSTI 4 immunization was carried out with synthetic heptapeptides, which comprise these target sites.Immunizations for the production of monoclonal antibodies can be performed both in vivo and in vitro. The advantages of the in vitro over in vivo immunization are (a) it requires only 5 -9 days, (b) the yield of positive stable hybridoma clones is considerably larger, (c) picomolar amounts of antigen are sufficient to produce monoclonal antibodies and (d) the probability of producing antibodies against self-antigens is greater, since suppression or tolerance mechanisms are less able to function. Nevertheless the poor survival capacity of

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