Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening.
The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS. A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E. coli transformant. As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein. Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography. The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa). The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins. The inhibitor could inhibit the gel softening of mackerel surimi.
Jiang
Shann-Tzong
ST
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China. sjiang@mail.ntou.edu.tw
Tzeng
Shinn-Shuenn
SS
Wu
Wun-Tsai
WT
Chen
Gen-Hung
GH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002645:Chickens / Q000235:genetics; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics*; D005396:Fish Products; D005503:Food Additives / Q000737:chemistry*; D005782:Gels / Q000737:chemistry*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics*
D015891:Cystatins; D005503:Food Additives; D005782:Gels; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins