A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background:The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.Methods:The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.Results:Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.Conclusions:The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)

Péptidos con actividad antimicrobiana producidos por microorfanismos nativos / Peptides with antimicrobial activity produced by isolated native microorganisms

Al igual que las proteínas, los péptidos antimicrobianos (PAM) son moléculas versátiles sintetizadas por microorganismos siguiendo rutas enzimáticas, y con interesantes propiedades alimentarias y farmacéuticas, entre ellas la capacidad antibiótica sobre patógenos. La búsqueda convencional de biomoléculas provenientes de microorganismos consiste en analizar químicamente su extracto crudo, lo cual es engorroso y prolongado. El método de aislamiento usado en esta investigación permitió localizar microorganismos con capacidad para producir PAM por interacción de cargas entre moléculas generadas en el metabolismo microbiano y un colorante cargado presente en el medio de cultivo. Se aislaron 20 muestras de suelos de varios pisos térmicos en medios selectivos. Se purificaron 35 cepas con base en la interacción con un colorante básico y se identificaron microorganismos del género Streptomyces sp. y Bacillus sp. Se obtuvieron proteínas de los extractos crudos de fermentaciones, identificando péptidos y aminoácidos por cromatografía de capa fina y electroforesis. Aquellos extractos con alto contenido proteico se evaluaron por bioautografía, y se encontraron 2 extractos de 35 con actividad inhibitoria sobre E. coli ATCC 8739 (halos de 8 mm). Se demuestra la efectividad del método para aislar y purificar microorganismos productores de péptidos cargados y que poseen actividad biológica e industrial de gran interés.

Like proteins, antimicrobial peptides (AMP) are versatile molecules synthesized by microorganisms using enzymatic pathways with no genetic code instruction. AMP have interesting properties in the food and pharmaceutical industries, like their antimicrobial ability against pathogens. Looking for biomolecules from microorganisms requires hard and time consuming chemical analysis of each microorganism extract. The microorganism isolation method proposed in this research allowed us to find antimicrobial peptides produced by bacteria, through interaction between a charged dye mixed with selective agar and metabolites produced by microorganisms. Twenty soil samples from different zones were isolated in selective media; thirty five strains were purified based on interaction between basic dye and charged molecules from bacteria. Streptomyces sp. y Bacillus sp. both genera were identified. Protein extracts were obtained from the isolated microorganisms cultivated in liquid media; peptides and amino acids were identified by thin layer chromatography and electrophoresis. Those extracts with high protein level were used to evaluate bioautography. Two extracts from 35 showed inhibitory activity against E. coli ATCC 8739 (8 mm halo). Method effectiveness for the isolation and the purifying of microorganisms able to produce charged molecules, of industrial interest is demonstrated.