Expression of Human Gelatinase B in Pichia pastoris Nita Roy,* Sriram Padmanabhan,* ,1 Mathew Smith,† ,2 Lihong Shi,† Marc Navre,† and Goutam Das* ,3 *Syngene Intl. Pvt. Ltd., Bangalore 561229, India; and †Affymax Research Institute, Santa Clara, California Received December 16, 1998, and in revised form March 12, 1999 Full-length human gelatinase B (FLGelB) and its C- terminal truncated form (dGelB) were expressed in Pichia pastoris strain GS115, using the Saccharomyces cerevisiae Mat  signal peptide. In both cases, a high level of the secreted protein could be detected by SDS– PAGE. The truncated gene was also expressed using the human gelatinase B native signal peptide. Secre- tion using the Mat  signal peptide was significantly greater than that from the native signal peptide. The recombinant products were purified and character- ized biochemically. The recombinant proteins, FLGelB and dGelB, were found to have similar bio- chemical properties and activity to that of the human gelatinase B native protein. © 1999 Academic Press Human gelatinase B (MMP-9) belongs to the family of matrix metalloproteases (MMPs) 4 (1). These en- zymes are involved in the breakdown of extracellular matrix components and are thought to play a critical role in tumor cell invasion, arthritis, periodontitis, and osteoporosis (2). All matrix metalloproteases have three definable structural domains of related primary sequences, including a propeptide domain, a catalytic NH 2 -terminal domain, and a hemopexin-like COOH domain (2). The MMPs (approx. 18 in number) are dependent on zinc for their activity and are secreted as inactive zymogens (3). The zymogens can be activated by proteinases or by organomercurial compounds (such as 4-amino phenylmercuric acetate) and proteolytic cleavage is usually accompanied by a reduction of mo- lecular weight of 10,000 Da or more (2). The latency of the proenzyme is due to a conserved cysteine that binds to zinc at the active center and it is the disrup- tion of this Cys–Zn 2+ interaction which results in the cleavage of an N-terminal propeptide fragment fol- lowed by self-degradation at the C-terminus in some cases (4). Different forms of human gelatinase B have been isolated from lung SV40-transformed fibroblasts (5) and other tumor cell lines such as HL-60, SK-N-SH, and MDA-MB-231 (6). Human gelatinase B has also been cloned and expressed in NSO myeloma cells (7), HT-1080 cells (8), HeLa cells (9), Escherichia coli (1, 10), and insect cells (11). In the present study we have cloned and expressed human gelatinase B (C-terminal truncated and full length) in Pichia pastoris. A C- terminal truncated form of the enzyme was expected to show similar activity to full-length enzyme, yet be more stable in a recombinant form based on similar studies with gelatinase A (7). The expression of human gelatinase B in P. pastoris obviates the disadvantages of low protein yields from mammalian cell culture as well as inhibition of the enzymes by the endogenous tissue inhibitor of metalloproteases in such expression systems. In P. pastoris, there is no aggregation of the recombinant gelatinase B, a problem present during expression in E. coli (12). Although, to the best of our knowledge, this is the first report where human gela- tinase B has been expressed in P. pastoris, a similar expression study of mouse gelatinase B in P. pastoris has been reported earlier (13). The data presented in this paper support the use of the P. pastoris expression system for the production of recombinant human gelatinase B which is structurally and functionally similar to the properties associated with the native gelatinase B. 1 Present address: Dr. Reddy’s Laboratory, Hyderabad 500034, India. 2 Present address: Strata Biosciences, Alameda, CA 94501. 3 To whom correspondence should be addressed at Syngene Intl. Pvt. Ltd., 20 Th Km Hosur Road, Hebbagodi, Bangalore District 561229, India. Fax: +91 80 842 2623. E-mail: goutam.das@ questintl.com. 4 Abbreviations used: MMP, matrix metalloprotease; FLGelB, full- length human gelatinase B; dGelB, C-terminal truncated form of human gelatinase B; APMA, 4-amino phenylmercuric acetate; YNBG, yeast nitrogen base without amino acids with glycerol; BMGY, buffered glycerol complex medium; BMMY, buffered metha- nol complex medium; SSC, sodium chloride–sodium citrate; WCW, wet cell weight. Protein Expression and Purification 16, 324 –330 (1999) Article ID prep.1999.1073, available online at http://www.idealibrary.com on 324 1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.