Bacterial expression strategies for human angiogenesis proteins.

We outline an expression strategy using Escherichia coli to obtain soluble components of a selected group of human proteins implicated in angiogenesis. These targets represent a heterogeneous group of proteins which for expression purposes were separated into cytoplasmic and helical membrane protein categories. Target selection was refined using a bioinformatic approach to generate a list of 50 experimental targets. A group consisting of forty-four cytoplasmic and signal-containing protein targets were amplified and cloned into multiple expression vectors. For this target category, we obtained 48% soluble expression products. In addition, we used a domain expression approach for six high molecular weight proteins predicted to contain membrane spanning helices to obtain soluble domain products. These results validate the utility of a bioinformatically driven high throughput approach to increase the number of soluble proteins or protein domains which can be used for multiple downstream applications.

Strategies for high-throughput gene cloning and expression.

High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.

Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.

Effect of thyroid status on thin-filament Ca2+ regulation and expression of troponin I in perinatal and adult rat hearts.

There is evidence for the existence of developmental changes in expression of troponin I (TNI) in cardiac thin filaments; however, regulation of TNI expression has not been described. We tested whether thyroid state affects expression of TNI using neonatal and adult rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Polyacrylamide gels of myofibrils from hearts of 7-, 14-, 21-, and 28-day-old animals indicated that both euthyroid and hypothyroid rats display a developmental shift toward the adult form of TNI. However, hypothyroid rats displayed a lower percentage of adult TNI at each age studied. When adult rats were made hypothyroid, the proportion of adult TNI decreased slightly. Thin-filament activity was determined from measurements of the effect of acidic pH on calcium activation of myofibrillar ATPase activity. Sensitivity to acidic pH was measured by the magnitude of shift in pCa50 (-log of half-maximally activating molar Ca2+) between pH 7.0 and 6.5. Euthyroid rats displayed developmental increases in pH sensitivity. At 7, 14, and 28 days of development, shifts in pCa50 were 0.11, 0.38, and 0.43 units, respectively. Hypothyroid rats displayed less pH sensitivity with pCa50 shifts of 0.07, 0.21, and 0.15 units at 7, 14, and 28 days of development. Adult hypothyroid rats displayed a 0.38-unit shift in pCa50, whereas euthyroid adults displayed a 0.44-unit shift. Our results indicate that pH sensitivity and expression of cardiac TNI are influenced by developmental stage and hormonal status.