Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm (“codon harmonization”) for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the “recoded” genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins

Non-Bulk-Like Solvent Behavior in the Ribosome Exit Tunnel

As nascent proteins are synthesized by the ribosome, they depart via an exit tunnel running through the center of the large subunit. The exit tunnel likely plays an important part in various aspects of translation. Although water plays a key role in many bio-molecular processes, the nature of water confined to the exit tunnel has remained unknown. Furthermore, solvent in biological cavities has traditionally been characterized as either a continuous dielectric fluid, or a discrete tightly bound molecule. Using atomistic molecular dynamics simulations, we predict that the thermodynamic and kinetic properties of water confined within the ribosome exit tunnel are quite different from this simple two-state model. We find that the tunnel creates a complex microenvironment for the solvent resulting in perturbed rotational dynamics and heterogenous dielectric behavior. This gives rise to a very rugged solvation landscape and significantly retarded solvent diffusion. We discuss how this non-bulk-like solvent is likely to affect important biophysical processes such as sequence dependent stalling, co-translational folding, and antibiotic binding. We conclude with a discussion of the general applicability of these results to other biological cavities

Improving the Physical Diagnosis Skills of Third-year Medical Students: A Controlled Trial of a Literature-based Curriculum

OBJECTIVE: To determine if a literature-based physical diagnosis curriculum could improve student knowledge, skill, and self-confidence in physical diagnosis. DESIGN: Prospective controlled trial of an educational intervention. SETTING: Required internal medicine clerkship for third-year medical students at Brown Medical School. PARTICIPANTS: Third-year medical students who completed the internal medicine clerkship during the academic year 1999–2000: 32 students at 1 clerkship site received the intervention; a total of 50 students at 3 other clerkship sites served as controls. INTERVENTION: Physical diagnosis curriculum based on 8 articles from the Journal of the American Medical Association's Rational Clinical Examination series. Intervention students met weekly for 1 hour with a preceptor to review each article, discuss the sensitivity and specificity of the maneuvers and findings, and practice the techniques with an inpatient who agreed to be visited and examined. MEASUREMENTS AND MAIN RESULTS: Physical diagnosis knowledge for the 8 topics was evaluated using a 22-item multiple choice question quiz, skill was evaluated using trained evaluators, and self-confidence was assessed using an end-of-clerkship survey. Intervention students scored significantly higher than the control group on the knowledge quiz (mean correct score 70% vs 63%, P = .002), skills assessment (mean correct score 90% vs 54%, P < .001), and self-confidence score (mean total score 40 vs 35, P = .003), and they expressed greater satisfaction with the physical diagnosis teaching they received in the clerkship. CONCLUSION: This physical diagnosis curriculum was successful in improving students' knowledge, skill, and self-confidence in physical diagnosis

The Effect of English Language Proficiency on Length of Stay and In-hospital Mortality

BACKGROUND: In ambulatory care settings, patients with limited English proficiency receive lower quality of care. Limited information is available describing outcomes for inpatients. OBJECTIVE: To investigate the effect of English proficiency on length of stay (LOS) and in-hospital mortality. DESIGN: Retrospective analysis of administrative data at 3 tertiary care teaching hospitals (University Health Network) in Toronto, Canada. PARTICIPANTS: Consecutive inpatient admissions from April 1993 to December 1999 were analyzed for LOS differences first by looking at 23 medical and surgical conditions (59,547 records) and then by a meta-analysis of 220 case mix groups (189,119 records). We performed a similar analysis for in-hospital mortality. MEASUREMENTS: LOS and odds of in-hospital death for limited English-proficient (LEP) patients relative to English-proficient (EP) patients. RESULTS: LEP patients stayed in hospital longer for 7 of 23 conditions (unstable coronary syndromes and chest pain, coronary artery bypass grafting, stroke, craniotomy procedures, diabetes mellitus, major intestinal and rectal procedures, and elective hip replacement), with LOS differences ranging from approximately 0.7 to 4.3 days. A meta-analysis using all admission data demonstrated that LEP patients stayed 6% (approximately 0.5 days) longer overall than EP patients (95% confidence interval, 0.04 to 0.07). LEP patients were not at increased risk of in-hospital death (relative odds, 1.0; 95% confidence interval, 0.9 to 1.1). CONCLUSIONS: Patients with limited English proficiency have longer hospital stays for some medical and surgical conditions. Limited English proficiency does not affect in-hospital mortality. The effect of communication barriers on outcomes of care in the inpatient setting requires further exploration, particularly for selected conditions in which length of stay is significantly prolonged