Protein engineering by expressed protein ligation.

By allowing the controlled assembly of synthetic peptides and recombinant polypeptides, expressed protein ligation permits unnatural amino acids, biochemical probes, and biophysical probes to be specifically incorporated into semisynthetic proteins. A powerful feature of the method is its modularity; once the reactive recombinant pieces are in hand and the optimal ligation conditions have been developed, it is possible to quickly generate an array of semisynthetic analogs by simply attaching different synthetic peptide cassettes--in most cases the synthetic peptides will be small and easy to make. From a practical perspective, the rate-determining step in the process is usually not the ligation step (it is based on a simple and efficient chemical reaction), but rather the generation of the reactive polypeptide building blocks. In particular, optimizing the yields of recombinant polypeptide building blocks can require some initial effort. However, it should be noted that the initial investment in time required to optimize the production of the recombinant fragment is offset by the ease and speed with which one can produce the material thereafter. In the example described in this chapter, the yield of soluble intein fusion protein was slightly better using the GyrA intein than for the VMA intein, although in both cases significant amounts of fusion protein were present in the cell pellet. Studies are currently underway to identify optimal refolding conditions for GyrA fusion proteins solubilized from inclusion bodies.

Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils.

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

Distinct isoforms of chicken decorin contain either one or two dermatan sulfate chains.

Decorin, a member of a family of proteins with leucine-rich repeat motifs, is a widely distributed extracellular matrix proteoglycan that is thought to be responsible for the structure, tissue organization, and surface properties of fibrils. In mammals, decorin carries one chondroitin/dermatan sulfate chain as a distinction from its homologue, biglycan, which contains two glycosaminoglycan chains. With the aim to study decorin-collagen interactions in chicken, where the fibrillar organization of cartilage collagens is best understood, we have isolated decorin-related proteoglycans from sternal cartilage of 40-day-old broiler chickens. Small chondroitin/dermatan sulfate proteoglycans were resolved by hydrophobic interaction chromatography into two fractions, DCN I and DCN II. Both forms contained dermatan sulfate and, in addition, keratan sulfate chains. Tryptic fingerprinting revealed that the core proteins of DCN I and DCN II were identical. The protein was identified as decorin by amino-terminal sequencing. DCN II was found to contain two dermatan sulfate chains, whereas DCN I had a single dermatan sulfate chain. The dermatan sulfate attachment sites are located near the NH2 terminus of the core protein, i.e. at Ser-4 and Ser-16 in DCN II and at Ser-4 in DCN I. The keratan sulfate attachment sites are located in the central portion of the core protein, at Asn-179 and Asn-230. The presence of two dermatan sulfate chains renders the chicken proteoglycan DCN II structurally similar to mammalian biglycan. Interestingly, biglycan has not been detected in chicken. Therefore, in birds, DCN II may function as a biglycan substitute.