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On May 24, 2019, the FDA granted regular approval to alpelisib in combination with fulvestrant for postmenopausal women, and men, with hormone receptor (HR)-positive, HER2-negative, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. Approval was based on the SOLAR-1 study, a randomized, double-blind, placebo-controlled trial of alpelisib plus fulvestrant versus placebo plus fulvestrant. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST v1.1 in the cohort of trial participants whose tumors had a PIK3CA mutation. The estimated median PFS by investigator assessment in the alpelisib plus fulvestrant arm was 11 months [95% confidence interval (CI), 7.5-14.5] compared with 5.7 months (95% CI, 3.7-7.4) in the placebo plus fulvestrant arm (HR, 0.65; 95% CI, 0.50-0.85; two-sided P = 0.001). The median overall survival was not yet reached for the alpelisib plus fulvestrant arm (95% CI, 28.1-NE) and was 26.9 months (95% CI, 21.9-NE) for the fulvestrant control arm. No PFS benefit was observed in trial participants whose tumors did not have a PIK3CA mutation (HR, 0.85; 95% CI, 0.58-1.25). The most common adverse reactions, including laboratory abnormalities, on the alpelisib plus fulvestrant arm were increased glucose, increased creatinine, diarrhea, rash, decreased lymphocyte count, increased gamma glutamyl transferase, nausea, increased alanine aminotransferase, fatigue, decreased hemoglobin, increased lipase, decreased appetite, stomatitis, vomiting, decreased weight, decreased calcium, decreased glucose, prolonged activated partial thromboplastin time, and alopecia., (©2020 American Association for Cancer Research.)

One of the main challenges in this post genomic era is the development and implementation of efficient methods of protein synthesis. A clear understanding of the role of genes in an organism is to comprehend the biological functions of all of its proteins. Acquiring this knowledge will depend in part on the success of rapid synthesis and purification of proteins. The future of structural genomics and functional proteomics depends on the availability of abundantly expressing, soluble proteins in a high-throughput manner. Conventional cell based methods of protein expression is rather laborious, time consuming and the ways to fail are numerous including solubility, toxicity to the host and instability (e.g. proteolysis). Cell-free or in vitro protein synthesis, on the other hand allows the expression and analysis of protein synthesis, may solve many of these problems. It is a simple open system which lends itself for manipulations and modifications to influence protein folding, disulfide bond formation, incorporation of unnatural amino acids, protein stability (by incorporating protease inhibitors in the system) and even the expression of toxic proteins. Cell-free synthesis can also be used as a reliable screening methodology for subsequent protein expression in vivo. Furthermore, this technology is readily amenable to automation. Here, we present a protocol for expressing recombinant proteins with high yield in a standard 96-well plate format using E. coli cell-free extract in a batch mode.