Directed evolution of proteins using the modified protein stability increased by directed evolution and the β-lactamase tripartite fusion systems. The test protein BPTI (shown in orange) is inserted into either the minor coat protein of the fd phage, g3p, (A) or β-lactamase (B), creating a tripartite fusion. (A) During phage replication in Escherichia coli, g3p is first synthesized as an integral membrane protein (shown) before it is assembled into the phage coat (not shown). The N-terminal domain of g3p carries the recognition site for the bacterial receptors the phage binds to during infection, and the C-terminal domain anchors g3p into the phage coat. Therefore, both domains must remain covalently linked for g3p to be functional and maintain phage infectivity. (B) β-lactamase (bla) is a periplasmic enzyme that inactivates β-lactam antibiotics. The ability of the tripartite fusions to confer phage infectivity (A) or resistance to antibiotics (B) is strongly dependent on the in vivo stability of the guest protein that is inserted into the reporter protein. If the test protein is stable and folded properly, the two halves of the reporter protein remain covalently linked and confer activity (left). If the inserted protein is not properly folded or unstable, it is prone to degradation by periplasmic proteases (depicted as scissors) and is likely to be degraded (right). This leads to a separation of the N- and C-terminal fragments of g3p/β-lactamase, resulting in a decreased phage titer/antibiotic resistance (13). BPTI, bovine pancreatic trypsin inhibitor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

Increased destabilization of the Cys14-Cys38 disulfide bond in vitro correlates with increased antibiotic resistance in the β-lactamase system in vivo. Different variants of BPTI were inserted into β-lactamase via flexible linkers. The level of antibiotic resistance for cells expressing the corresponding fusion constructs was determined as described in the Materials and Methods section. BPTI variants containing single amino acid substitutions that were either identified in the phage selection (A16T, F33L, Y35N, and Y35G) or obtained from the literature (Y35L) are indicated with filled diamonds (17). The relative level of antibiotic resistance (indicated by MIC) compared to a tripartite fusion containing BPTI WT in vivo is plotted against the thermodynamic destabilization ΔΔG of the Cys14-Cys38 disulfide bond in the mutant proteins as determined in vitro (ΔΔG = R T ln[C_eff (WT)/C_eff(mut)], where C_eff (WT) and C_eff (mut) are the effective concentrations of the Cys14-Cys38 disulfide bond in WT BPTI or the mutant proteins, respectively) (17). Stability values for a BPTI species in which the formation of Cys14-Cys38 was prevented by either carboxymethylation or carboxyamidomethylation were taken from Schwarz et al. (36) and plotted against the level of resistance conferred by bla′-BPTI C14S C38S-′bla. These latter variants are proteins with chemical side chain modifications rather than mutations, and their thermodynamic destabilization values were determined using a different methodology. Therefore, their absolute values may not be precisely comparable to the single amino acid substitutions; they are shown as open diamonds. The R² value for the single point variants (filled diamonds) is 0.89, and the R² for the complete data set is 0.91. Data are expressed as mean values ± SD. MIC, minimal inhibitory concentration; SD, standard deviation.

The majority of BPTI [14–38] detected in whole cell extracts is periplasmic, but insoluble. Cell fractions were prepared from E. coli MG1655b ΔampC cells expressing bla′-BPTI WT-′bla or bla′-BPTI [14–38]-′bla, respectively, as described in the Material and Methods section. The tripartite fusions were detected by Western blot using an anti-β-lactamase antibody. In cells treated with sodium azide, the Sec pathway is blocked and the export of the tripartite fusions prevented (NaN₃). Here, tripartite fusion proteins remain in the cytosol and retain their signal sequence, which is usually cleaved upon export in the periplasm. Tripartite fusions that were exported (as detected in the periplasmic extracts [PE], indicated with peri.) can therefore be distinguished from fusion proteins that were retained in the cytosol (as detected in the sample treated with NaN₃, indicated with cyto.) by their molecular weight. The blot shows that for both bla′-BPTI WT-′bla and bla′-BPTI [14-38]-′bla, all fusion protein detected in whole cell extracts (WC) is located in the periplasm. However, for bla′-BPTI [14-38]-′bla the amount of soluble protein is much lower than for bla′-BPTI WT-′bla (compare soluble [SO] and insoluble fractions [IS]).

Substitution of Cys17 in hG-CSF with serine leads to an increase in antibiotic resistance as part of the β-lactamase system. WT hG-CSF, which contains two disulfide bonds (Cys36-Cys42 and Cys64-Cys74) and one unpaired cysteine (Cys17), is prone to aggregation, even under native-like conditions (35). Substitution of the free cysteine with serine significantly decreases the proteins propensity to aggregate (35). Mid-log phase cells of E. coli MG1655 ΔampC expressing bla′-hG-CSF WT-′bla or bla′-hG-CSF C17S-′bla, respectively, were normalized to OD_{600 nm} = 1. Serial dilutions of cultures from 10⁰ to 10⁻⁵ were spotted on LB plates containing different concentrations of ampicillin. After 18 h of incubation at 37°C, growth or no growth for each dilution for each ampicillin concentration was determined.

Insertion of BPTI into β-lactamase leads to a massive drop in antibiotic resistance compared to cells expressing the unmodified enzyme. Mid-log phase cells of E. coli MG1655 ΔampC expressing TEM1-β-lactamase (bla WT) or TEM1-β-lactamase fused with WT BPTI (bla′-BPTI WT-′bla), respectively, were normalized to OD_600 nm = 1. Serial dilutions of cultures from 10⁰ to 10⁻⁵ were spotted on LB plates containing different concentrations of ampicillin. After 18 h of incubation at 37°C, growth or no growth for each dilution for each ampicillin concentration was determined. WT, wild-type.

In BPTI variants that can only form a single disulfide bond, the stability of the disulfide bond in vitro correlates with the level of antibiotic resistance in the β-lactamase system in vivo. (A) Three variants of BPTI representing single disulfide-bonded intermediates ([14–38], [30–51], or [5–55], respectively) were inserted into β-lactamase via flexible linkers. In all variants, the cysteines not involved in formation of the disulfide bond indicated were substituted with serines. Bla WT indicates WT β-lactamase without any insertions, and BPTI WT refers to the construct bla-′BPTI WT-′bla. The level of antibiotic resistance (indicated by MIC) for cells expressing the corresponding fusion constructs was determined as described in the Materials and Methods section and is normalized to cells expressing bla′-BPTI WT-′bla. (B) The “effective concentration” of two interacting groups in the native-like conformation can be used as an expression of the free energy of the intermolecular interaction (23). The higher the effective concentration, the larger is the contribution of the particular interaction to the stability of the folded formation. Values for the effective concentrations were taken from Creighton and Goldenberg (7). (C) In the reduced state, the kinetic effective concentration can be used to assess the extent to which two cysteines are brought into proximity to each other (9). Values for the effective concentration for the three disulfide bonds indicated were taken from Dadlez and Kim (9). Data are expressed as mean values ± SD.

Steady-state protein levels of different bla′-BPTI-′bla variants in vivo correlate well with the corresponding levels of antibiotic resistance. The relative steady-state protein levels in whole cell extracts were detected by Western blot using an anti-β-lactamase antibody. Band intensities of β-lactamase (circle) or tripartite fusions containing different BPTI variants (filled diamonds) were normalized to the protein levels of cells expressing bla′-BPTI WT-′bla (open circle). In the case of bla′-BPTI [14–38]′-bla, the protein level was determined in whole cell extracts (open diamond) and in soluble, periplasmic fractions (triangle). In both cases, the protein level was adjusted to the protein level of bla′-BPTI WT′-bla in whole cell extracts or the soluble, periplasmic cell fraction, respectively. The trend line includes the value for the soluble periplasmic fraction of bla′-BPTI [14–38]′-bla and excludes the value for the whole cell extract of bla′-BPTI [14–38]′-bla. Data are expressed as mean values ± SD.

Consensus folding pathway of WT BPTI in vitro, modified from Creighton and Goldenberg (7) and Weissman and Kim (8, 45, 46). Starting from fully reduced BPTI (far left), the [14–38] intermediate is formed much more rapidly than any other species containing one disulfide-bond (9). However, the vast majority of [14–38] molecules are either reduced or isomerized before they form the thermodynamically more stable intermediates [30–51] or [5–55] (1). The most productive folding pathway for BPTI proceeding from the single disulfide-bonded species occurs via the two intermediates [5–14; 30–51] and [5–38; 30–51], which are in rapid equilibrium with each other and contain non-native disulfides (5, 10). At neutral pH, a large fraction of the BPTI molecules accumulates as kinetic traps (N′ and N*), which only slowly isomerize toward the very native-like species (45, 46). Once is formed, the Cys14-Cys38 disulfide is introduced rapidly to obtain native BPTI (4). Note that the relative distribution of the double disulfide-bonded species is dependent on the pH (10, 45). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).