A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli.
Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin.
Yasukawa
Kiyoshi
K
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Kusano
Masayuki
M
Inouye
Kuniyo
K
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013820:Thermolysin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002364:Caseins; D011994:Recombinant Proteins; D013820:Thermolysin; D000409:Alanine