This work is part of a retrospective collection of 179 electronic theses and dissertations (ETDs) from the VCU Libraries pilot ETD system that were designated as available only to VCU users. Please contact us at if you have questions or if you are the author of one of these and would like to release it for online public access.

Non-VCU users: Please talk to your librarian about requesting this thesis through interlibrary loan.

Defense Date


Document Type


Degree Name

Master of Science



First Advisor

Dr. H T Wright


The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.


Part of Retrospective ETD Collection, restricted to VCU only.


© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008