Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, ...and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.
•GFP nanobody was expressed and purified using prokaryotic and eukaryotic expression systems.•Purified GFP nanobody can be used to identify GFP protein and GFP-tagged protein.•The GFP nanobodies derived from two protein expression systems were found to have different GFP recognition abilities.•MS analysis found that GFP nanobodies from two protein expression systems have different post-translational modifications, which may be a promising development trend for nanobody research.
Aim/Hypothesis: Obesity is associated with increased triacylglycerol (TAG) storage in adipose tissue and insulin resistance. The mobilization of stored TAG is mediated by hormone-sensitive lipase ...(HSL) and the recently discovered adipose triglyceride lipase (ATGL). The aim of the present study was to examine whether ATGL and HSL mRNA and protein expression are altered in insulin-resistant conditions. In addition, we investigated whether a possible impaired expression could be reversed by a period of weight reduction.
Methods: Adipose tissue biopsies were taken from obese subjects (n = 44) with a wide range of insulin resistance, before and just after a 10-wk hypocaloric diet. ATGL and HSL protein and mRNA expression was determined by Western blot and quantitative RT-PCR, respectively.
Results: Fasting insulin levels and the degree of insulin resistance (using the homeostasis model assessment index for insulin resistance) were negatively correlated with ATGL and HSL protein expression, independent of age, gender, fat cell size, and body composition. Both mRNA and protein levels of ATGL and HSL were reduced in insulin-resistant compared with insulin-sensitive subjects (P < 0.05). Weight reduction significantly decreased ATGL and HSL mRNA and protein expression. A positive correlation between the decrease in leptin and the decrease in ATGL protein level after weight reduction was observed. Finally, ATGL and HSL mRNA and protein levels seem to be highly correlated, indicating a tight coregulation and transcriptional control.
Conclusions: In obese subjects, insulin resistance and hyperinsulinemia are strongly associated with ATGL and HSL mRNA and protein expression, independent of fat mass. Data on weight reduction indicated that also other factors (e.g. leptin) relate to ATGL and HSL protein expression.
Escherichia coli
is the most widely used heterologous protein expression system. However, this system remains a challenge due to the low solubility of proteins, insufficient yield, and inclusion body ...formation. Numerous approaches have sought to address these issues. The use of a fusion tag is one of the most powerful strategies for obtaining large amounts of heterologous protein in
E. coli
expression system
.
Here, recent advances in fusion tags that increase the expression of proteins are reviewed. In addition, proposed concepts for designing peptide tags to increase protein expression are discussed.
PDF
