The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
A xylanase DNA sequence with a total length of 642 bp was previously isolated from a xylanolytic Klebsiellapneumoniae. Xylanase gene primers were designed with the addition of BamH1 and EcoR1 restriction enzymesites in order get a full xylanase gene that is in-frame with pSTAG expression vector. The isolated xylanasegene was amplified using the designed primers through PCR, then cloned and expressed in E. coli BL21 (DE3).In-silico characterization showed that the recombinant xylanase has a molecular weight of 23.9 kDa and a pI of9.32. The signal peptide cleavage site for the recombinant xylanase was predicted to be between residues 61and 62. The activity of the crude recombinant xylanase was 2.015 U/mL, which was higher than the crudenative xylanase activity, with maximum at 0.642 U/mL. Staining of the birchwood xylan agar plate with Congored showed a clearing zone around E. coli BL21 (DE3) colonies with recombinant pSTAG plasmid evenwithout being induced with IPTG. This implied leaky expression of the E. coli BL21 (DE3) secretion system,which recognized the signal sequence of the recombinant xylanase, and proceeded to cleave and secreted outthe mature protein into the culture medium. MALDI-TOF analysis of a 20 kDa protein present in the culturemedium confirmed that the recombinant xylanase had been secreted into the culture medium.
Background Gel staining is a crucial step that allows the visualisation of proteins separated through SDS-PAGE. Colloidal Coomassie Brilliant Blue-G (CBB-G) staining is among the commonly used visualisation methods due to several factors such as compatibility with mass spectrometry (MS) analysis, sensitivity, reproducibility, and simplicity of the staining process. However, the standard colloidal CBB-G staining has a drawback: the resolution of protein bands is compromised because of diffusion of proteins during the washing step. Results A modification to an established colloidal CBB-G staining method, which greatly increases the resolution of protein bands, is described. The addition of a fixation step, which prevents the diffusion of proteins during the washing step, is shown to increase protein band resolution. Conclusion The fixation step is fast, flexible, and also retains all the advantages of the standard colloidal CBB-G staining methods. As there are no drawbacks, incorporating this fixation step into the standard colloidal CBB-G staining is an easy way to improve protein visualisation in SDS-PAGE. Graphical Abstract
Piper nigrum L., also known as the pepper plant, produces pepper berries, which is a spice used worldwide. Changes in the natural environment have forced the plant to undergo acclimatization for plant growth. Consequently, un-adapted plants developed unhealthy, stunted growth, affecting the production of pepper berries, and thus, causing loss to agriculture. Proteomic analysis using two-dimensional polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry was used to compare the differentially expressed proteins between healthy and unhealthy pepper plants. The proteins were successfully extracted from the stems and roots of P. nigrum and the two-dimensional protein profiles were compared. Two-dimensional analysis has indicated significant differences between the healthy and unhealthy parts with 41 and 25 differentially expressed proteins in the stems and roots respectively. A total of 10 differentially expressed proteins, characterized as unique (2) and over-expressed (8) proteins, from both plant parts were selected and sent for identification through matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry. The unique and over-expressed proteins in the unhealthy stems were identified as stress-related small heat shock proteins (sHSPs). These proteins act as a chaperone to protect other proteins against a stressful environment for plant defence and adaptation. Meanwhile, over-expressed proteins from the roots were found to be peroxidase 5, malate dehydrogenase, mitochondrial and gamma-glutamyl phosphate reductase which are involved in oxidative stress, photosynthetic activity, and plant development, respectively. The findings of the differentially expressed proteins in this study, therefore, may create opportunities for further investigations of stress management control and help to improve plantation practices in the future.
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