A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

The Influence of the Nucleotide Composition of Genes and Gene Regulatory Elements on the Efficiency of Protein Expression in Escherichia coli.

Recombinant proteins expressed in Escherichia coli are widely used in biochemical research and industrial processes. At the same time, achieving higher protein expression levels and correct protein folding still remains the key problem, since optimization of nutrient media, growth conditions, and methods for induction of protein synthesis do not always lead to the desired result. Often, low protein expression is determined by the sequences of the expressed genes and their regulatory regions. The genetic code is degenerated; 18 out of 20 amino acids are encoded by more than one codon. Choosing between synonymous codons in the coding sequence can significantly affect the level of protein expression and protein folding due to the influence of the gene nucleotide composition on the probability of formation of secondary mRNA structures that affect the ribosome binding at the translation initiation phase, as well as the ribosome movement along the mRNA during elongation, which, in turn, influences the mRNA degradation and the folding of the nascent protein. The nucleotide composition of the mRNA untranslated regions, in particular the promoter and Shine-Dalgarno sequences, also affects the efficiency of mRNA transcription, translation, and degradation. In this review, we describe the genetic principles that determine the efficiency of protein production in Escherichia coli.

Fragmentation of human cardiac troponin T after acute myocardial infarction.

BACKGROUND: Blood measurement of cardiac troponin T (cTnT) is one of the most widespread methods of acute myocardial infarction (MI) diagnosis. cTnT degradation may have a significant influence on the precision of cTnT immunodetection; however, there are no consistent data describing the level and sites of cTnT proteolysis in the blood of MI patients. In this study, we bordered major cTnT fragments and quantified their relative abundance in the blood at different times after MI. METHODS: Serial heparin plasma samples were collected from 37 MI patients 2-37 h following the onset of MI. cTnT and its fragments were studied by western blotting and immunofluorescence analysis using monoclonal antibodies specific to various cTnT epitopes. RESULTS: cTnT was present in the blood of MI patients as 23 proteolytic fragments with an apparent molecular mass of âˆ¼ 8-37 kDa. Two major sites of cTnT degradation were identified: between amino acid residues (aar) 68 and 69 and between aar 189 and 223. Analysis of the abundance of cTnT fragments showed an increase in the fraction of free central fragments in the first few hours after MI, while the fraction of the C-terminal fragments of cTnT remained almost unchanged. CONCLUSION: cTnT progressively degrades after MI and appears in the blood as a mixture of 23 proteolytic fragments. The cTnT region approximately bordered by aar 69-158 is a promising target for antibodies used for measurement of total cTnT.