Quantificação de erros de incorporação em proteínas recombinantes

Mestrado em Biologia Molecular CelularA síntese de proteínas de acordo com o código genético é essencial para manter proteoma estável e para a homeostase celular. No entanto, os erros podem ocorrer naturalmente durante a síntese da proteína a partir do seu mRNA, variando entre 10-3 a 10-4 erros por codão. Estes erros ocorrem com mais frequncia em proteínas recombinantes sobre-expressas em hospedeiros heterólogos. Quantidades crescentes de proteína não-funcional estão geralmente relacionados à tradução em condições de stress. Neste estudo utilizou-se Saccharomyces cerevisiae como um organismo hospedeiro para expressar o gene lacZ-GST para quantificar o erro de tradução. A levedura foi tratada com diversos agentes de stress tais como o etanol, o crómio (CrO3), e aminoglicósido antibiótico - geneticina (G418). A incorporação de erros foi estudada em proteína solúvel e insolúvel para determiner se os erros de tradução aumentam a agregação de proteína. Usando esta abordagem, verificou-se que o stress aumenta o erro de tradução para níveis de 5.6 × 10-3 a 8 × 10-3, 60 - 80 vezes mais que o nível normal. Esta taxa de erro inesperadamente elevada tem implicações para a utilização terapêutica de proteínas recombinantes.The synthesis of protein according to genetic code of a gene determines the basis of life and a stable proteome is necessary for cell homeostatis. Faithful translation of protein give gurantee of cell survival. However, errors occur naturally during translation of protein from its mRNA, which varies from 10-3 to 10-4 per codon. These errors are more frequent in recombinant protein overexpressed in heterologous hosts and affect protein functionality. The increasing amount of nonfunctional protein is often related to mistranslation of a gene under stress. In the present study, we used Saccharomyces cerevisiae as a host organism to overexpress E. coli lacZ gene fusion with GST to quantify misincorporation of amino acid in GST-β galactosidase recombinant protein. The yeast was treated with various stressors such as ethanol, chromium (CrO3), and aminoglycoside antibiotic - geneticin (G418) to induce protein aggregation. The misincorporation of amino acids was studied in both soluble and insoluble protein fractions by mass-spectrometry to determine how much misincorporation occur and whether this is associated with protein insolubility. We found that under experimental stress conditions the misincorporation of amino acids ranges from 5.6 × 10-3 to 8 × 10-3, which represents 60-80 fold higher than reported level. The unexpectedly high error rate has implications for the therapeutic use of the heterologous host derived recombinant proteins

Molecular evolution of a genetic code alteration

Doutoramento em BiologiaDurante os últimos anos, foram descritas alterações ao código genético, quer em procariotas, quer em eucariotas, quebrando o dogma de que o código genético é universal e imutável. Estudos recentes sugerem que a evolução de tais alterações requerem modificações ao nível da estrutura da maquinaria da tradução e são promovidas por mecanismos de descodificação ambígua. Em C. albicans, um organismo que é patogénico para o Homem, a alteração ao código genético é mediada por uma alteração na estrutura de um novo tRNACAG de serina que descodifica o codão CUG de leucina como serina. De forma a determinar se este tRNA, que é aminoacilado pelas Seryl- e Leucyl- tRNA sintetases, promove a descodificação ambígua do codão CUG, foi desenvolvido um sistema para a quantificar in vivo, por espectrometria de massa, os níveis de incorporação de serina e de leucina em codões CUG. Os resultados mostraram que em condições normais de crescimento leucina é incorporada a uma taxa de 3% e que serina é incorporada a uma taxa de 97%. No entanto, o nível de ambiguidade na descodificação de codões CUG aumentou para 5% em células crescidas em condições de stress, indicando que a incorporação de leucina em codões CUG é sensível a factores ambientais e é manipulada durante a tradução do mRNA. Tal, levanta a hipótese de que a incorporação de leucina poderá atingir níveis superiores aos determinados neste estudo. Para testar esta hipótese e determinar os níveis máximos de ambiguidade na descodificação do codão CUG tolerados pelas células, aumentou-se artificialmente a ambiguidade do codão CUG em C. albicans. Surpreendentemente, a incorporação de leucina subiu de 5% para 28%, o que representa um aumento na taxa de erro da tradução de 3500 vezes, relativamente ao descrito para o mecanismo de tradução. Dado existirem 13.000 codões CUG no genoma de C. albicans, a sua descodificação ambígua expande de uma forma exponencial o proteoma deste fungo, criando assim um proteoma estatístico, resultante da síntese de um conjunto de moléculas diferentes para cada proteína a partir de um único RNA mensageiro (mRNA) que contenha codões CUG. Os resultados obtidos demonstraram que o proteoma de C. albicans tem uma dimensão muito superior à prevista pelo seu genoma e demonstram um papel central da descodificação ambígua na evolução do código genético.Alterations to the standard genetic code have been found in both prokaryotes and eukaryotes, demolishing the dogma of an immutable and universal genetic code. Recent studies suggest that evolution of such alterations require structural change of the translation machinery and are driven through mechanisms that require codon decoding ambiguity. In the human pathogen C. albicans, a structural change in a novel sertRNACAG allows for its recognition by both the LeuRS and SerRS in vitro and in vivo, providing such molecular device. In order to determine whether this tRNA charging ambiguity results in ambiguous CUG decoding, we have developed a system for quantification of the level of serine and leucine at the CUG codon by Mass-Spectrometry. The data showed that 3.0% of leucine and 97.0% of serine are incorporated at CUG codons in vivo under standard growth conditions. Moreover, this ambiguity increases up to 5.0% under stress, indicating that it is sensitive to environmental change and raising the hypothesis that leucine incorporation may be higher than determine experimentally. In order to determine the scope of C. albicans tolerance to CUG ambiguity, we have created highly ambiguous C. albicans cell lines through tRNA engineering. These cell lines tolerated up to 28% leucine incorporation at CUGs, which represents an increase of 3500 fold in decoding error rate. Since there are 13,000 CUG codons in C. albicans such ambiguity expands the proteome exponentially and creates a statistical proteome due to synthesis of arrays of protein molecules from mRNAs containing CUG codons. The overall data showed that the dimension of the C. albicans proteome is far higher than that predicted from its genome and provides important new evidence for a pivotal role for codon ambiguity in the evolution of the genetic code

Engineering the glycoside hydrolase β-glucuronidase (β-GUS) to improve a non-native activity for the synthesis of O-glucuronides

The demand for O-glucuronides as potential therapeutic products and biomarkers continue to increase. However, large-scale synthesis of O glucuronides remains a challenge for the industry, and has prompted the development of an alternative synthetic routes. This dissertation extends from a previous enzyme engineering work that had introduced a site-specific mutation E504G in β glucuronidase (β-GUS), resulting in a functional glucuronylsynthase (Syn). However, the synthetic activity of Syn is low and leaves ample scope for improvement. The work described in this thesis aims to produce a more efficient glucuronylsynthase using different enzyme engineering approaches. Two separate strategies were employed to achieve our objective. The first strategy engages a two-step process where the β-GUS is first engineered to have higher activity in the presence of excess substrate; 10–20 times its Km. This is followed by site-specific mutation E504G to convert the β-GUS variant into a Syn. The second strategy engineers the glucuronylsynthase directly. Chapter 3 describes the attempt to improve the activity of the native enzyme in the presence of t-BuOH, a solvent that was found to improve the chemistry of the glucuronylsynthase chemoenzymatic reaction. The engineering attempt produced a potential variant with a mutation at its C-terminal region, L561S, that is more active in the presence of the solvent. This mutation appears to be a determinant mutation. Biophysical characterization of the enzyme revealed that this improvement is not due to increased stability in t-BuOH, while our analysis of the crystal structure suggests that the mutation improved the activity by increasing loop flexibility at the C-terminal region. Subsequently, I incorporated E504G into the β-GUS variant, but this did not translate into a better glucuronylsynthase variant. Chapter 4 describes the second strategy. Two mutations, H162Q and Y160G, at the N-terminal region were found to boost the synthetic activity but this was not accompanied by improvement in their thermostability nor solvent stability. However, combining the results from the biophysical characterization experiments and observations from the structural examination on 3K4D, it can be inferred that the mutations promote glucuronylsynthase activity by modulating the active site of the Syn so that it would favour the glucuronyl donor substrate. Therefore, these mutations would serve as concrete starting points for further evolution program of the Syn. Chapter 5 explores the potential reason that could account for the lack of success in transposing the potency of L561S to the glucuronylsynthase system. The work here is driven by the hypothesis that translational misincorporation introduced contaminating wild-type during enzyme expression. Essentially, this chapter highlights the potential pitfall of the glucuronylsynthase system and describes potential strategies to avoid this pitfall. Finally, Chapter 6 builds upon the results from β GUS engineering and explores the mutational tolerance of β GUS. Its mutational tolerance is compared with another enzyme that is structurally less complex (β-lactamase, TEM-1). In addition, its mutational tolerance with different substrate is also compared. This exercise attempts to provide insights into the elements that would drive the adaption process of the β-GUS. Consequently, we expect this study to facilitate future directed evolution studies of the β-GUS and the glucuronylsynthase